Plant transcription factor that is involved in sugar signalling

ABSTRACT

Provided are nucleotide sequences which encodes a sugar-signalling transcription factors capable of activating a promoter of a gene encoding an enzyme involved in the synthesis or deposition of starch e.g. in response to sugar levels in a plant. Preferred sequences encode WRKY proteins such as the sugar signaling in barley2 (SUSIBA2) factor and variants thereof, and act on promoters which comprise at least one SURE element and\or W box element to which the transcription factor binds (e.g. promoters such as iso1, sbe1, sbeIIb, ssI, agpaseS). The invention also provides related methods and materials e.g. for activating, modulating, and investigating the elements present in such promoters.

TECHNICAL FIELD

The present invention relates generally to methods and materials basedon transcription factors involved in sugar signalling in plants.

BACKGROUND ART

Development of the cereal seed is orchestrated by the coordinatedactivities of a large number of genes that encode metabolic andregulatory enzymes, as well as other proteins (Olsen, 2001). Thisresults in a triploid endosperm, the embryo, pericarp, seed coat andother tissues of the mature grain. The endosperm structure consists oftwo tissues, the interior starch-filled endosperm and the outerepidermal layer called the aleurone.

Starch, which is a mixture of amylopectin (a heavily branchedpolyglucan) and amylose (a mostly linear polyglucan), is deposited inthe endosperm as granules. The synthesis and deposition of starch in theendosperm depend on enzymes such as ADP-glucose pyrophosphorylase(AGPase), starch synthases (SS), starch branching enzymes (SBE), andstarch debranching enzymes (DBE). Most, if not all, of these enzymesexist in two or more isoforms (see Ball et al., 1998; Buleon et al.,1998; Myers et al., 2000; Nakamura, 2002; Smith, 2001; for recentreviews on starch biosynthesis). DBEs are grouped into two distinctclasses, isoamylase and pullulanase each with different isoforms(Nakamura, 1996). Isoamylase (EC 3.2.1.68) is an essential enzyme inamylopectin synthesis. However, the precise role of the enzyme in thisprocess is still not clear, and different models have been proposed(Ball et al., 1998; Nakamura, 2002; Smith, 2001).

It has been reported that expression of starch synthesis genes, i.e. SSin potato (Visser et al., 1991), ADPGase in potato (Müller-Röber et al.,1990), sweet potato (Bae and Liu, 1997), Arabidopsis (Rook et al., 2001)and tomato (Li et al, 2002), SBE in potato (Kossman et al., 1991), maize(Kim and Guiltinan, 1999) and Arabidopsis (Khoshnoodi et al., 1998), andisoamylase in barley (Sun et al., 1999), are sugar inducible. Incontrast to the situation in bacteria, yeast and mammals, where sugarsignaling cascades are extensively studied, the sugar signalingtransduction pathways in plants are poorly understood (Rolland et al.,2002). Generally, in higher plants, high sugar levels stimulateexpression of genes involved in sink function, such as growth, storageof proteins and biosynthesis of starch and other carbohydrates, whereaslow sugar levels promote photosynthesis and mobilization of energyreserves, such as breakdown of storage starch or lipids.

Sugar signaling can be dissected into three steps, sugar sensing, signaltransduction, and target gene expression. The picture is clouded by thedual function of sugars as nutrients and signaling molecules, and by theinteraction (in plants and animals) between sugar signaling and hormonalnetworks. In plants the complexity is further increased by the vitalrole of sugar production through photosynthesis. Hexoses, sucrose andtrehalose might serve as elicitors of plant sugar signaling (Goddijn andSmeekens, 1998; Rolland et al., 2002). Hexokinase, sucrose and glucosetransporters, and various sugar receptors, have been proposed ascomponents of the sugar sensing machinery (Rolland et al., 2002; Sheenet al., 1999; Smeekens, 2000).

The Ser/Thr protein kinase Snf1 is a central participant in yeast sugarsignaling (Carlson, 1999). Snf1 phosphorylates downstream components andis also itself activated by phosphorylation. Snf related protein kinases(SnRKs) are found in yeast, mammals and plants, where they partake in alarge number of regulatory functions (Halford and Hardie, 1998; Hardieet al., 1998). There is evidence that some plant SnRKs share functionalhomology with Snf1 in plant sugar signaling, although the exact natureof their responses to sugars remains to be clarified (Rolland et al.,2002). Other players implicated in sugar signaling transduction pathwaysin plants are sugar metabolites, 14-3-3 proteins, trehalose-6-phosphate,and Ca²⁺ (Rolland et al., 2002).

Little is known about the cis and trans factors mediating the finalsteps in plant sugar signaling. To date, five different types of ciselements have been identified in sugar-regulated plant promoters; theSURE (Grierson et al., 1994), SP8 (Ishiguro and Nakamura, 1994), TGGACGG(Maeo et al., 2001), G box (Giuliano et al., 1988) and B box (Griersonet al., 1994; Zourelidou et al., 2002) elements.

Only two putative transcription factors, SPF1 and STK, with relevance toplant sugar signaling have been isolated. SPF1 was isolated from sweetpotato, and it was reported that SPF1 binds to the SP8a and SP8bpromoter elements of the β-amylase and sporamin genes of tuberous roots,where it functions as a repressor (Ishiguro and Nakamura, 1994). STKbinds to the B box as an activator (Zourelidou et al., 2002).

It can thus be seen that novel transcription factors, for instance thosewhich could be used to affect starch synthesis or deposition (and hencestructure) may provide a useful contribution to the art.

DISCLOSURE OF THE INVENTION

The present inventors have isolated a transcription factor, SUSIBA2(sugar signaling in barley), and examined its interaction with endospermspecific expression of the sbeIIb (encoding SBEIIb) and iso1 (encodingisoamylase 1) genes during barley seed development.

Antibodies against SUSIBA2 were produced and the expression pattern forsusiba2 was determined at the RNA and protein levels. It was found thatsusiba2 is expressed in endosperm but not leaves. Transcription ofsusiba2 is sugar inducible and ectopic susiba2 expression was obtainedin sugar-treated leaves. The temporal expression of susiba2 in barleyendosperm followed that of iso1 and endogenous sucrose levels, with apeak at around 12 days after pollination.

It was previously known that barley iso1 harbors an SP8a element andthat it contributes to the endosperm specificity of iso1 expression byrecruiting a repressor in non-expressing tissues (Sun et al., 1999).Similarly, the endosperm-specific expression of barley sbeIIb (Sun etal., 1998) is partly determined by a repressor-binding B box element,Bbl, in the second intron of the gene (Ahlandsberg et al., 2002b).

Interestingly SUSIBA2 appears to bind, as an activator, to the SURE(sugar responsive) element in the iso1 promoter. It also binds the W boxelements but not to the SP8a element. A novel application of atranscription factor oligodeoxynucleotide decoy strategy withtransformed barley endosperm was used to provide experimental evidencefor the importance of the SURE element in iso1 transcription.

Thus, not only is its biding specificity different to SPF1 and STKdescribed above, but SUSIBA2 also represents the first isolatedSURE-binding transcription factor, the first WRKY protein known to beinvolved in carbohydrate anabolism, and the first transcription factorof any sort reported for regulation of starch synthesis. It shares onlya very low degree of sequence identity (28%) with SPF1. Orthologs toSUSIBA2 were also isolated from rice and wheat endosperm.

The sequences from the rice and wheat cDNAs, and wheat peptide areannexed hereto.

Thus in a first aspect of the invention there is disclosed an isolatednucleic acid which comprises, or consists essentially of, a nucleotidesequence which encodes a transcription factor which is capable ofmodulating the activity of a promoter of a gene encoding an enzymeinvolved in the synthesis or deposition of starch.

“Nucleic acid” and “nucleic acid molecule” have the same meaning. Asstated above, the nucleic acid may consist essentially of a nucleotidesequence of the present invention (which is to say that the sequence is‘of the essence of’ the molecule, generally making up more than 50% ofit).

The promoter will preferably include at least one SURE element (asdescribed in more detail hereinafter—see Example 7 in particular) and\orW box element and the transcription factor will bind to one or more ofthese.

The enzyme (and hence promoter) may optionally be selected from thefollowing genes: iso1, sbe1, sbeIIb, ssI, agpaseS.

Preferably the transcription factor is a WRKY protein which is involvedin carbohydrate anabolism. More specifically it modulates the activityof the promoter within a plant in response to sugar levels in the planti.e. the factor is a component of a sugar signaling pathway which isresponsible for modulating target gene expression.

Preferably the nucleotide sequence is a SUSIBA2 nucleotide sequencewhich encodes the 573-amino acid sequence given in FIG. 1.

Preferably the nucleotide sequence is a SUSIBA2 nucleotide sequencewhich consists of the coding sequence given in FIG. 1 or one which isdegeneratively equivalent or complementary thereto. The length of thesusiba2 cDNA is 2355 nucleotides, and the open reading frame starts atposition 247 and ends at position 1966 (FIG. 1).

In alternative embodiments the nucleotide sequence is a SUSIBA2nucleotide sequence which encodes an amino acid sequence given in theSequence Annex. In preferred alternative embodiments the nucleotidesequence is a SUSIBA2 nucleotide sequence which comprises a codingsequence given in the Sequence Annex or one which is degenerativelyequivalent or complementary thereto. Where aspects or embodiments of theinvention are discussed in relation to the barley sequence given in FIG.1, it will be understood that each of those aspects or embodimentsapplies mutatis mutandis to the wheat or rice orthologues, as describedin the Sequence Annex.

The nucleic acid molecules or vectors (see below) according to thepresent invention may be provided isolated and/or purified from theirnatural environment, in substantially pure or homogeneous form, or freeor substantially free of nucleic acid or genes of the species ofinterest or origin other than the sequence encoding a polypeptide withthe required function. The term “isolated” encompasses all thesepossibilities.

Nucleic acid according to the present invention may include cDNA, RNA,genomic DNA and modified nucleic acids or nucleic acid analogs. Where aDNA sequence is specified, e.g. with reference to a figure, unlesscontext requires otherwise the RNA equivalent, with U substituted for Twhere it occurs, is encompassed.

Complement sequences of those discussed herein are also encompassed. Asis well understood by those skilled in the art, two nucleic acidnucleotide sequences are “complementary” when one will properly basepair with all or part of the other according to the standard rules (Gpairs with C, and A pairs with T). One sequence is “the complement” ofanother where those sequences are of the same length, but arecomplementary to each other.

In a further aspect of the present invention there is disclosed anucleic acid molecule comprising (or preferably consisting essentiallyof) a nucleotide sequence which is a variant of the barley SUSIBA2nucleotide sequence of FIG. 1.

Thus the invention provides, inter alia, an isolated transcriptionfactor gene derivable from (and which can be expressed recombinantly in)maturing seeds, and which encodes a transcription factor protein whichtargets a promoter of a gene encoding an enzyme involved in thesynthesis or deposition (anabolism) of starch. Such genes may encodeSUSIBA2, or are related or derived therefrom e.g. genes which encode anRNA which hybridizes to the SUSIBA2 gene under high stringencyconditions. The transcription factors may bind a SURE and\or W boxelement in the promoter, and optionally not bind the SP8a element.

The gene may be present in an expression cassette, and the transcriptionfactor of the invention may be used, for example, in a method forenhancing or reducing expression of a starch anabolic enzyme, whichmethod may involve transforming a seed crop plant with the factor gene,said plant thereby expressing the transcription factor protein encodedby said transcription factor gene. This may be used to give enhanced,reduced, or altered starch synthesis. More specifically the disclosureof these sequences provides a novel mechanism for manipulating thestarch anabolism activity in plants in a number of important respects.These include, inter alia, the ability to: modulate the activity ofisoamylases and other enzymes in plants; alter responsiveness of thispathway to sugar levels; alter overall debranching enzyme activity;alter such enzyme activities in various different tissues or subcellularcompartments or at various different developmental stages and producenovel starch types in transgenic line.

Some aspects and embodiments of the invention will now be discussed inmore detail.

SUSIBA2 Variants

Variants of the present invention can be artificial nucleic acids (i.e.containing sequences which have not originated naturally) which can beprepared by the skilled person in the light of the present disclosure.Artificial variants (derivatives) may be prepared by those skilled inthe art, for instance by site directed or random mutagenesis, or bydirect synthesis. Preferably the variant nucleic acid is generatedeither directly or indirectly (e.g. via one or amplification orreplication steps) from an original nucleic acid having all or part ofthe sequences of the first aspect. Preferably the variant encodes aproduct which has one or more of the transcription factor activitiesdiscussed above.

Alternatively they may be novel, naturally occurring, nucleic acids,isolatable using the sequences of the present invention. Sequencevariants which occur naturally may also include alleles (which willinclude polymorphisms or mutations at one or more bases). Preferrednucleic acids are those encoding orthologs from any of rice, wheat, orpotato or derivatives of these as described in more detail below.

Thus a variant may be or include a distinctive part or fragment (howeverproduced) corresponding to a portion of the sequence provided. Theseportions may include motifs which are distinctive to SUSIBA2 sequences,such motifs being discussed in more detail in the Examples below.

Fragments may encode or omit particular functional parts of thepolypeptide. Equally the fragments may have utility in probing for, oramplifying, the sequence provided or closely related ones. Suitablelengths of fragment, and conditions, for such processes are discussed inmore detail below. Also included are nucleic acids which have beenextended at the 3′ or 5′ terminus with respect to those of the firstaspect.

The term “variant” nucleic acid as used herein encompasses all of thesepossibilities. When used in the context of polypeptides or proteins itindicates the encoded expression product of the variant nucleic acid.

Some of the aspects of the present invention relating to variants willnow be discussed in more detail.

Homology (either similarity or identity) may be as defined anddetermined by the TBLASTN program, of Altschul et al. (1990) J. Mol.Biol. 215: 403-10, which is in standard use in the art, or, and this maybe preferred, the standard program BestFit, which is part of theWisconsin Package, Version 8, September 1994, (Genetics Computer Group,575 Science Drive, Madison, Wis., USA, Wisconsin 53711). BestFit makesan optimal alignment of the best segment of similarity between twosequences. Optimal alignments are found by inserting gaps to maximizethe number of matches using the local homology algorithm of Smith andWaterman.

Homology, with respect to SUSIBA2 in FIG. 1 may be at the nucleotidesequence and/or encoded amino acid sequence level. Preferably, thenucleic acid and/or amino acid sequence shares at least about 50%, or60%, or 70%, or 80% homology, most preferably at least about 90%, 95%,96%, 97%, 98% or 99% similarity or identity.

Thus a variant polypeptide in accordance with the present invention mayinclude within an amino acid sequences described herein a single aminoacid or 2, 3, 4, 5, 6, 7, 8, or 9 changes, about 10, 15, 20, 30, 40 or50 changes, or greater than about 50, 60, 70, 80, 90, 100 or 115changes. In addition to one or more changes within the amino acidsequence shown, a variant polypeptide may include additional amino acidsat the C-terminus and/or N-terminus.

Thus in a further aspect of the invention there is disclosed a method ofproducing a derivative nucleic acid comprising the step of modifying thecoding sequence of a nucleic acid comprising any one the sequencesdiscussed above.

Changes to a sequence, to produce a derivative, may be by one or more ofaddition, insertion, deletion or substitution of one or more nucleotidesin the nucleic acid, which may lead to the addition, insertion, deletionor substitution of one or more amino acids in the encoded polypeptide.Changes may be desirable for a number of reasons, including introducingor removing the following features: restriction endonuclease sequences;codon usage; other sites which are required for post translationmodification; cleavage sites in the encoded polypeptide; motifs in theencoded polypeptide (e.g. binding sites). Leader or other targetingsequences (e.g. the putative TM region) may be added or removed from theexpressed protein to determine its location following expression. All ofthese may assist in efficiently cloning and expressing an activepolypeptide in recombinant form (as described below).

Other desirable mutation may be random or site directed mutagenesis inorder to alter the activity (e.g. specificity) or stability of theencoded polypeptide. Changes may be by way of conservative variation,i.e. substitution of one hydrophobic residue such as isoleucine, valine,leucine or methionine for another, or the substitution of one polarresidue for another, such as arginine for lysine, glutamic for asparticacid, or glutamine for asparagine. As is well known to those skilled inthe art, altering the primary structure of a polypeptide by aconservative substitution may not significantly alter the activity ofthat peptide because the side-chain of the amino acid which is insertedinto the sequence may be able to form similar bonds and contacts as theside chain of the amino acid which has been substituted out. This is soeven when the substitution is in a region which is critical indetermining the peptides conformation. Also included are variants havingnon-conservative substitutions. As is well known to those skilled in theart, substitutions to regions of a peptide which are not critical indetermining its conformation may not greatly affect its activity becausethey do not greatly alter the peptide's three dimensional structure.

In regions which are critical in determining the peptides conformationor activity such changes may confer advantageous properties on thepolypeptide. Indeed, changes such as those described above may conferslightly advantageous properties on the peptide e.g. altered stabilityor specificity.

Other methods for generating novel specificities may include mixing orincorporating sequences from related SUSIBA genes into the sequencesdisclosed herein. For example restriction enzyme fragments of relatedgenes could be ligated together. An alternative strategy for modifyingSUSIBA2 sequences would employ PCR as described below (Ho et al., 1989,Gene 77, 51-59) or DNA shuffling (Crameri et al., 1998, Nature 391).

In a further aspect of the present invention there is provided a methodof detecting, identifying and/or cloning (isolating) a nucleic acid ofthe present invention (e.g. a homologue of the sequences set outhereinafter) from a plant which method employs any of the sequences ofthe invention discussed above. In particular the methods will generallyemploy primers or probes derived from all or part of these sequences (orsequences complementary thereto) set out herein.

An oligonucleotide primer for use in amplification reactions may beabout 30 or fewer nucleotides in length. Generally specific primers areupwards of 12, 13, 14, 15, 18, 21 or 24 nucleotides in length. Foroptimum specificity and cost effectiveness, primers of 16-24 nucleotidesin length may be preferred.

An oligonucleotide or polynucleotide probe may be based on the any ofthe sequences disclosed herein (e.g. introns or exons, although thelatter may be preferred). If required, probing can be done with entirerestriction fragments of the genes which may be 100's or even 1000's ofnucleotides in length.

Those skilled in the art are well versed in the design of primers foruse processes such as PCR. The primers will usually be based onsequences which are peculiar or unique to the SUSIBA2 sequences (seee.g. discussion of C-terminal sequence in the Examples below).

When using such probes or primers, if need be, clones or fragmentsidentified in the search can be extended. For instance if it issuspected that they are incomplete, the original DNA source (e.g. aclone library, mRNA preparation etc.) can be revisited to isolatemissing portions e.g. using sequences, probes or primers based on thatportion which has already been obtained to identify other clonescontaining overlapping sequence.

In one embodiment, a variant in accordance with the present invention isalso obtainable by means of a method which includes:

(a) providing a preparation of nucleic acid, e.g. from plant cells,

(b) providing a probe or primer as discussed above,

(c) contacting nucleic acid in said preparation with said nucleic acidmolecule under conditions for hybridisation of said nucleic acidmolecule to any said gene or homologue in said preparation, andidentifying said gene or homologue if present by its hybridisation withsaid nucleic acid molecule.

Plants which may be a suitable source of SUSIBA2 may include any cereal,and in particular the developing seeds of such cereals.

Probing may employ the standard Southern blotting technique. Forinstance DNA may be extracted from cells and digested with differentrestriction enzymes. Restriction fragments may then be separated byelectrophoresis on an agarose gel, before denaturation and transfer to anitrocellulose filter. Labelled probe may be hybridised to the DNAfragments on the filter and binding determined. DNA for probing may beprepared from RNA preparations from cells.

Test nucleic acid may be provided from a cell as genomic DNA, cDNA orRNA, or a mixture of any of these, preferably as a library in a suitablevector. If genomic DNA is used the probe may be used to identifyuntranscribed regions of the gene (e.g. promoters etc.), such as isdescribed hereinafter. Probing may optionally be done by means ofso-called “nucleic acid chips” (see Marshall & Hodgson (1998) NatureBiotechnology 16: 27-31, for a review).

Preliminary experiments may be performed by hybridising under lowstringency conditions. For probing, preferred conditions are those whichare stringent enough for there to be a simple pattern with a smallnumber of hybridisations identified as positive which can beinvestigated further.

For instance, screening may initially be carried out under conditions,which comprise a temperature of about 37° C. or less, a formamideconcentration of less than about 50%, and a moderate to low salt (e.g.Standard Saline Citrate (“SSC”)=0.15 M sodium chloride; 0.15 M sodiumcitrate; pH 7) concentration.

Alternatively, a temperature of about 50° C. or less and a high salt(e.g. “SSPE” 0.180 mM sodium chloride; 9 mM disodium hydrogen phosphate;9 mM sodium dihydrogen phosphate; 1 mM sodium EDTA; pH 7.4). Preferablythe screening is carried out at about 37° C., a formamide concentrationof about 20%, and a salt concentration of about 5×SSC, or a temperatureof about 50° C. and a salt concentration of about 2×SSPE. Theseconditions will allow the identification of sequences which have asubstantial degree of homology (similarity, identity) with the probesequence, without requiring the perfect homology for the identificationof a stable hybrid.

Suitable conditions include, e.g. for detection of sequences that areabout 80-90% identical, hybridization overnight at 42° C. in 0.25MNa₂HPO₄, pH 7.2, 6.5% SDS, 10% dextran sulfate and a final wash at 55°C. in 0.1×SSC, 0.1% SDS. For detection of sequences that are greaterthan about 90% identical, suitable conditions include hybridizationovernight at 65° C. in 0.25M Na₂HPO₄, pH 7.2, 6.5% SDS, 10% dextransulfate and a final wash at 60° C. in 0.1×SSC, 0.1% SDS.

It is well known in the art to increase stringency of hybridisationgradually until only a few positive clones remain. Suitable conditionswould be achieved when a large number of hybridising fragments wereobtained while the background hybridisation was low. Using theseconditions nucleic acid libraries, e.g. cDNA libraries representative ofexpressed sequences, may be searched. Those skilled in the art are wellable to employ suitable conditions of the desired stringency forselective hybridisation, taking into account factors such asoligonucleotide length and base composition, temperature and so on.

Binding of a probe to target nucleic acid (e.g. DNA) may be measuredusing any of a variety of techniques at the disposal of those skilled inthe art. For instance, probes may be radioactively, fluorescently orenzymatically labelled. Other methods not employing labelling of probeinclude amplification using PCR (see below) or RNAase cleavage. Theidentification of successful hybridisation is followed by isolation ofthe nucleic acid which has hybridised, which may involve one or moresteps of PCR or amplification of a vector in a suitable host.

In a further embodiment, hybridisation of nucleic acid molecule to avariant may be determined or identified indirectly, e.g. using a nucleicacid amplification reaction, particularly the polymerase chain reaction(PCR). PCR requires the use of two primers to specifically amplifytarget nucleic acid, so preferably two nucleic acid molecules withsequences characteristic of are employed. Using RACE PCR, only one suchprimer may be needed (see “PCR protocols; A Guide to Methods andApplications”, Eds. Innis et al, Academic Press, New York, (1990)).

Thus a method involving use of PCR in obtaining nucleic acid accordingto the present invention may be carried out as described above, butusing a pair of nucleic acid molecule primers useful in (i.e. suitablefor) PCR.

The methods described above may also be used to determine the presenceof one of the nucleotide sequences of the present invention within thegenetic context of an individual plant, optionally a transgenic plantwhich may be produced as described in more detail below. This may beuseful in plant breeding programmes e.g. to directly select plantscontaining alleles which are responsible for desirable traits in thatplant species, either in parent plants or in progeny (e.g. hybrids, F1,F2 etc.). Thus use of the markers defined in the Examples below, ormarkers which can be designed by those skilled in the art on the basisthe nucleotide sequence information disclosed herein, forms one part ofthe present invention.

As used hereinafter, unless the context demands otherwise, the term“SUSIBA2 nucleic acid” is intended to cover any of the nucleic acids ofthe invention described above, including functional variants.

SUSIBA2 Vectors and Transformation

In one aspect of the present invention, the SUSIBA2 nucleic aciddescribed above is in the form of a recombinant and preferablyreplicable vector.

“Vector” is defined to include, inter alia, any plasmid, cosmid, phageor Agrobacterium binary vector in double or single stranded linear orcircular form which may or may not be self transmissible or mobilizable,and which can transform prokaryotic or eukaryotic host either byintegration into the cellular genome or exist extrachromosomally (e.g.autonomous replicating plasmid with an origin of replication).

Specifically included are shuttle vectors by which is meant a DNAvehicle capable, naturally or by design, of replication in two differenthost organisms, which may be selected from actinomycetes and relatedspecies, bacteria and eucaryotic (e.g. higher plant, yeast or fungalcells).

A vector including nucleic acid according to the present invention neednot include a promoter or other regulatory sequence, particularly if thevector is to be used to introduce the nucleic acid into cells forrecombination into the genome.

Preferably the nucleic acid in the vector is under the control of, andoperably linked to, an appropriate promoter or other regulatory elementsfor transcription in a host cell such as a microbial, e.g. bacterial, orplant cell. The vector may be a bi-functional expression vector whichfunctions in multiple hosts. In the case of genomic DNA, this maycontain its own promoter or other regulatory elements and in the case ofcDNA this may be under the control of an appropriate promoter or otherregulatory elements for expression in the host cell

By “promoter” is meant a sequence of nucleotides from whichtranscription may be initiated of DNA operably linked downstream (i.e.in the 3′ direction on the sense strand of double-stranded DNA).

“Operably linked” means joined as part of the same nucleic acidmolecule, suitably positioned and oriented for transcription to beinitiated from the promoter. DNA operably linked to a promoter is “undertranscriptional initiation regulation” of the promoter.

Thus this aspect of the invention provides a gene construct, preferablya replicable vector, comprising a promoter operatively linked to anucleotide sequence provided by the present invention.

Generally speaking, those skilled in the art are well able to constructvectors and design protocols for recombinant gene expression. Suitablevectors can be chosen or constructed, containing appropriate regulatorysequences, including promoter sequences, terminator fragments,polyadenylation sequences, enhancer sequences, marker genes and othersequences as appropriate. For further details see, for example,Molecular Cloning: a Laboratory Manual: 2nd edition, Sambrook et al,1989, Cold Spring Harbor Laboratory Press.

Many known techniques and protocols for manipulation of nucleic acid,for example in preparation of nucleic acid constructs, mutagenesis (seeabove discussion in respect of variants), sequencing, introduction ofDNA into cells and gene expression, and analysis of proteins, aredescribed in detail in Current Protocols in Molecular Biology, SecondEdition, Ausubel et al. eds., John Wiley & Sons, 1992. The disclosuresof Sambrook et al. and Ausubel et al. are incorporated herein byreference.

In one embodiment of this aspect of the present invention, there isprovided a gene construct, preferably a replicable vector, comprising aninducible promoter operatively linked to a nucleotide sequence providedby the present invention.

The term “inducible” as applied to a promoter is well understood bythose skilled in the art. In essence, expression under the control of aninducible promoter is “switched on” or increased in response to anapplied stimulus. The nature of the stimulus varies between promoters.Some inducible promoters cause little or undetectable levels ofexpression (or no expression) in the absence of the appropriatestimulus. Other inducible promoters cause detectable constitutiveexpression in the absence of the stimulus. Whatever the level ofexpression is in the absence of the stimulus, expression from anyinducible promoter is increased in the presence of the correct stimulus.

Particular of interest in the present context are nucleic acidconstructs which operate as plant vectors. Specific procedures andvectors previously used with wide success upon plants are described byGuerineau and Mullineaux (1993) (Plant transformation and expressionvectors. In: Plant Molecular Biology Labfax (Croy RRD ed) Oxford, BIOSScientific Publishers, pp 121-148).

Suitable promoters which operate in plants include the CauliflowerMosaic Virus 35S (CaMV 35S). Other examples are disclosed at pg 120 ofLindsey & Jones (1989) “Plant Biotechnology in Agriculture” Pub. OUPress, Milton Keynes, UK. The promoter may be selected to include one ormore sequence motifs or elements conferring developmental and/ortissue-specific regulatory control of expression. Inducible plantpromoters include the ethanol induced promoter of Caddick et al (1998)Nature Biotechnology 16: 177-180.

If desired, selectable genetic markers may be included in the construct,such as those that confer selectable phenotypes such as resistance toantibiotics or herbicides (e.g. kanamycin, hygromycin, phosphinotricin,chlorsulfuron, methotrexate, gentamycin, spectinomycin, imidazolinonesand glyphosate).

The present invention also provides methods comprising introduction ofsuch a construct into a host cell, particularly a plant cell.

In a further aspect of the invention, there is disclosed a host cellcontaining a heterologous nucleic acid or construct according to thepresent invention, especially a plant or a microbial cell.

The term “heterologous” is used broadly in this aspect to indicate thatthe SUSIBA2 nucleic acid in question has been introduced into said cellsof the plant or an ancestor thereof, using genetic engineering, i.e. byhuman intervention. A heterologous gene may replace an endogenousequivalent gene, i.e. one which normally performs the same or a similarfunction, or the inserted sequence may be additional to the endogenousgene or other sequence.

Nucleic acid heterologous to a plant cell may be non-naturally occurringin cells of that type, variety or species. Thus the heterologous nucleicacid may comprise a coding sequence of or derived from a particular typeof plant cell or species or variety of plant, placed within the contextof a plant cell of a different type or species or variety of plant. Afurther possibility is for a nucleic acid sequence to be placed within acell in which it or a homolog is found naturally, but wherein thenucleic acid sequence is linked and/or adjacent to nucleic acid whichdoes not occur naturally within the cell, or cells of that type orspecies or variety of plant, such as operably linked to one or moreregulatory sequences, such as a promoter sequence, for control ofexpression.

The host cell (e.g. plant cell) is preferably transformed by theconstruct, which is to say that the construct becomes established withinthe cell, altering one or more of the cell's characteristics and hencephenotype e.g. with respect to sugar sensing or starch anabolism.

Nucleic acid can be transformed into plant cells using any suitabletechnology, such as a disarmed Ti-plasmid vector carried byAgrobacterium exploiting its natural gene transfer ability (EP-A-270355,EP-A-0116718, NAR 12(22) 8711-87215 1984), particle or microprojectilebombardment (U.S. Pat. No. 5,100,792, EP-A-444882, EP-A-434616)microinjection (WO 92/09696, WO 94/00583, EP 331083, EP 175966, Green etal. (1987) Plant Tissue and Cell Culture, Academic Press),electroporation (EP 290395, WO 8706614 Gelvin Debeyser) other forms ofdirect DNA uptake (DE 4005152, WO 9012096, U.S. Pat. No. 4,684,611),liposome mediated DNA uptake (e.g. Freeman et al. Plant Cell Physiol.29: 1353 (1984)), or the vortexing method (e.g. Kindle, PNAS U.S.A. 87:1228 (1990d) Physical methods for the transformation of plant cells arereviewed in Oard, 1991, Biotech. Adv. 9: 1-11.

Agrobacterium transformation is widely used by those skilled in the artto transform dicotyledonous species. Recently, there has also beensubstantial progress towards the routine production of stable, fertiletransgenic plants in almost all economically relevant monocot plants(see e.g. Hiei et al. (1994) The Plant Journal 6, 271-282)).Microprojectile bombardment, electroporation and direct DNA uptake arepreferred where Agrobacterium alone is inefficient or ineffective.Alternatively, a combination of different techniques may be employed toenhance the efficiency of the transformation process, eg bombardmentwith Agrobacterium coated microparticles (EP-A-486234) ormicroprojectile bombardment to induce wounding followed byco-cultivation with Agrobacterium (EP-A-486233).

The particular choice of a transformation technology will be determinedby its efficiency to transform certain plant species as well as theexperience and preference of the person practising the invention with aparticular methodology of choice. It will be apparent to the skilledperson that the particular choice of a transformation system tointroduce nucleic acid into plant cells is not essential to or alimitation of the invention, nor is the choice of technique for plantregeneration.

Thus a further aspect of the present invention provides a method oftransforming a plant cell involving introduction of a construct asdescribed above into a plant cell and causing or allowing recombinationbetween the vector and the plant cell genome to introduce a nucleic acidaccording to the present invention into the genome.

The invention further encompasses a host cell transformed with nucleicacid or a vector according to the present invention especially a plantor a microbial cell. In the transgenic plant cell (i.e. transgenic forthe nucleic acid in question) the transgene may be on an extra-genomicvector or incorporated, preferably stably, into the genome. There may bemore than one heterologous nucleotide sequence per haploid genome.

Generally speaking, following transformation, a plant may beregenerated, e.g. from single cells, callus tissue or leaf discs, as isstandard in the art. Almost any plant can be entirely regenerated fromcells, tissues and organs of the plant. Available techniques arereviewed in Vasil et al., Cell Culture and Somatic Cell Genetics ofPlants, Vol I, II and III, Laboratory Procedures and Their Applications,Academic Press, 1984, and Weissbach and Weissbach, Methods for PlantMolecular Biology, Academic Press, 1989.

The generation of fertile transgenic plants has been achieved in thecereals rice, maize, wheat, oat, and barley (reviewed in Shimamoto, K.(1994) Current Opinion in Biotechnology 5, 158-162; Vasil, et al. (1992)Bio/Technology 10, 667-674; Vain et al., 1995, Biotechnology Advances 13(4): 653-671; Vasil, 1996, Nature Biotechnology 14 page 702).

Plants which include a plant cell according to the invention are alsoprovided.

In addition to the regenerated plant obtainable by the above method, thepresent invention embraces all of the following: a clone of such aplant; selfed or hybrid progeny; descendants (e.g. F1 and F2descendants) and any part of any of these. The invention also provides aplant propagule from such plants, that is any part which may be used inreproduction or propagation, sexual or asexual, including cuttings, andso on. In each case these embodiments will include a heterologousSUSIBA2 nucleic acid according to the present invention.

Uses of SUSIBA2 Nucleic Acids and Transcription Factors

The present invention provides a method of influencing the starchanabolism in a plant, the method including the step of causing orallowing expression of the product (polypeptide or nucleic acidtranscript) encoded by heterologous nucleic acid according to theinvention from that nucleic acid within cells of the plant.

The step may be preceded by the earlier step of introduction of thenucleic acid into a cell of the plant or an ancestor thereof.

Also provided is a method of binding, activating, or identifying apromoter which includes at least one SURE element and\or W box element,which method employs the step of contacting said promoter with atranscription factor as described herein. Where such method is performedin vivo, it will be a heterologous transcription factor.

Activation of a promoter may be confirmed by use of a reporter gene e.g.as described in the Examples herein.

Also provided is a method of identifying a sugar responsive element in apromoter, which method employs the step of contacting said promoter witha transcription factor as described herein. Where such method isperformed in vivo, it will generally be a heterologous transcriptionfactor. The contacting step is optionally followed by detection ofbinding to the promoter or region of the promoter. For example, elementswithin promoters may be characterised by use of EMSA or DNAasefoot-printing techniques e.g. as described in the Examples herein.

The foregoing discussion has been generally concerned with uses of thenucleic acids of the present invention for production of functionalSUSIBA2 polypeptides in a plant.

However the information disclosed herein may also be used to reduce theactivity or levels of such polypeptides in cells in which it is desiredto do so. For instance the sequence information disclosed herein may beused for the down-regulation of expression of genes e.g. usinganti-sense technology (see e.g. Bourque, (1995), Plant Science 105,125-149); sense regulation [co-suppression] (see e.g. Zhang et al.,(1992) The Plant Cell 4, 1575-1588). Further options for down regulationof gene expression include the use of ribozymes, e.g. hammerheadribozymes, which can catalyse the site-specific cleavage of RNA, such asmRNA (see e.g. Jaeger (1997) “The new world of ribozymes” Curr OpinStruct Biol 7:324-335. Nucleic acids and associated methodologies forcarrying out down-regulation (e.g. complementary sequences) form onepart of the present invention. As is well known to those skilled in theart, Double stranded RNA (dsRNA) has been found to be even moreeffective in gene silencing than both sense or antisense strands alone(Fire A. et al Nature, Vol 391, (1998)). dsRNA mediated silencing isgene specific and is often termed RNA interference (RNAi) (See also Fire(1999) Trends Genet. 15: 358-363, Sharp (2001) Genes Dev. 15: 485-490,Hammond et al. (2001) Nature Rev. Genes 2: 1110-1119 and Tuschl (2001)Chem. Biochem. 2: 239-245).

The present invention also encompasses the expression product of any ofthe functional nucleic acid sequences disclosed above, plus also methodsof making the expression product by expression from encoding nucleicacid therefore under suitable conditions, which may be in suitable hostcells.

Following expression, the recombinant product may, if required, beisolated from the expression system. Generally however the polypeptidesof the present invention will be used in vivo (in particular in planta).

Modified Starches

The present inventors have confirmed the importance of SUSIBA2 inregulation of starch synthesis by use of antisense oligodeoxynucleotide(ODN) technology (Dagle, J. M. & Weeks, D. L., (2001) Differentiation69, 75-82). The ectopic expression of iso1 and sbeIIb was followed inintersected sucrose-treated barley leaves after addition of an 18-merantisense ODN against susiba2. RNA-gel blot analysis revealed thatexposure to the antisense ODN suppressed iso1 and sbeIIb expression,providing functional evidence that SUSIBA2 is relevant for iso1 andsbeIIb activities.

Additionally the inventors investigated the effects of the antisense ODNtechnology on starch composition. The antisense susiba2 ODNs lead to asignificant increase in iodine staining, which is indicative of fewer orshorter branches in the starch molecules, consistent with reduced sbeIIband iso1 activities. Also the solubility properties of the starchappeared to be altered.

Thus as discussed hereinbefore, the present invention provides a methodof modifying polysaccharide (e.g. starch) production in a plant, themethod including the step of down-regulating the expression of SUSIBA2in the plant. Most preferably the down regulation comprises use of anoligonucleotide (ODN) based technology e.g. comprises the step ofintroducing antisense ODN into the plant, which antisense ODN comprisesall or part of the complementary SUSIBA2 sequence e.g.5′-CGCGGGGGACATGGCCTT-3′ as described below. Preferred methodologies forreducing gene expression using oligonucleotides may be any of thosereviewed by Dagle, J. M. & Weeks, D. L., (2001) supra.

In a further aspect of the present invention there is provided apolysaccharide generated (in vivo) by a process comprising manipulationof a SUSIBA2 transcription factor as described above. Also embraced isstarch produced in the transformed plants and cells discussed above.Such starch is preferably derived from amylopectin but has any of adecreased, increased or otherwise altered degree of branching, with acorresponding alteration in properties e.g. solubility, swelling orability to form a paste rather than a gel when heated in water.Commodities (e.g. foodstuffs) comprising such starches form a furtheraspect of the present invention.

Other commodities which may benefit from the modified starches of thepresent invention include biodegradable plastics; food-processingthickeners; starch coated films, papers & textiles; paint thickeners;mining explosives; pharmaceuticals and glues. The modified starches canbe used analogously to prior art starches in these materials, in wayswhich are well known to those skilled in the respective technicalfields.

SUSIBA2 Antibodies

Purified SUSIBA2 protein produced recombinantly by expression fromencoding nucleic acid therefor, may be used to raise antibodiesemploying techniques which are standard in the art. Antibodies andpolypeptides comprising antigen-binding fragments of antibodies form afurther part of the present invention, and may be used in identifyinghomologues from other plant species.

Methods of producing antibodies include immunising a mammal (e.g. mouse,rat, rabbit, horse, goat, sheep or monkey) with the protein or afragment thereof. Antibodies may be obtained from immunised animalsusing any of a variety of techniques known in the art, and might bescreened, preferably using binding of antibody to antigen of interest.

For instance, Western blotting techniques or immunoprecipitation may beused (Armitage et al, 1992, Nature 357: 80-82). Antibodies may bepolyclonal or monoclonal.

Antibodies may be modified in a number of ways. Indeed the term“antibody” should be construed as covering any polypeptide having abinding domain with the required SUSIBA2 specificity. Thus, this termcovers antibody fragments, derivatives, functional equivalents andhomologues of antibodies, including any polypeptide comprising animmunoglobulin binding domain, whether natural or synthetic. Chimaericmolecules comprising an immunoglobulin binding domain, or equivalent,fused to another polypeptide are therefore included. Cloning andexpression of Chimaeric antibodies are described in EP-A-0120694 andEP-A-0125023. It has been shown that fragments of a whole antibody canperform the function of binding antigens. Examples of binding fragmentsare (i) the Fab fragment consisting of VL, VH, CL and CH1 domains; (ii)the Fd fragment consisting of the VH and CH1 domains; (iii) the Fvfragment consisting of the Vl and VH domains of a single antibody; (iv)the dAb fragment (Ward, E. S. et al., Nature 341, 544-546 (1989) whichconsists of a VH domain; (v) isolated CDR regions; (vi) F(ab′)2fragments, a bivalent fragment comprising two linked Fab fragments (vii)single chain Fv molecules (scFv), wherein a VH domain and a VL domainare linked by a peptide linker which allows the two domains to associateto form an antigen binding site (Bird et al, Science, 242, 423-426,1988; Huston et al, PNAS USA, 85, 5879-5883, 1988); (viii) bispecificsingle chain Fv dimers (PCT/US92/09965) and (ix) “diabodies”,multivalent or multispecific fragments constructed by gene fusion(WO94/13804; P Holliger et al Proc. Natl. Acad. Sci. USA 90 6444-6448,1993).

Candidate SUSIBA2 polypeptides may be screened using theseantibodies—e.g. by screening the products of an expression librarycreated using nucleic acid derived from an plant of interest, or theproducts of a purification process from a natural source. A polypeptidefound to bind the antibody may be isolated and then may be subject toamino acid sequencing. Any suitable technique may be used to sequencethe polypeptide either wholly or partially (for instance a fragment ofthe polypeptide may be sequenced). Amino acid sequence information maybe used in obtaining nucleic acid encoding the polypeptide, for instanceby designing one or more oligonucleotides (e.g. a degenerate pool ofoligonucleotides) for use as probes or primers in hybridization tocandidate nucleic acid, or by searching computer sequence databases, asdiscussed further below.

The invention will now be further described with reference to thefollowing non-limiting Figures and Examples. Other embodiments of theinvention will occur to those skilled in the art in the light of these.

FIGURES

FIG. 1. Structure of the susiba2 cDNA from barley.

The 18-nucleotides long sequence complementary to the human IGF1receptor gene is underlined. The amino acid sequence of the trypticfragment obtained by microsequencing from the overproduced SUSIBA2 (seeFIG. 3) is shown by an open box.

FIG. 2. Analysis of the SUSIBA2 Primary Structure.

The amino acid sequence of SUSIBA2 was aligned with those of the nineclosest matching proteins from a BLAST search. Identical amino acids areshown in back boxes, and similar amino acids are shown in grey boxes.The WRKYGQK peptide stretch is shown in pink. Putative nuclearlocalization signals are indicated with an orange # sign under thesequences. Ser- and Thr-rich regions are underlined in red. Thezinc-finger-like motifs in the two WRKY domains are indicated with greenvertical arrows under the sequence. A. t. hyp, Arabidopsis hypotheticalprotein; A. t. put, Arabidopsis putative protein; A. t. unk, Arabidopsisunknown protein; A. t. wrky20, Arabidopsis WRKY transcription factor 20;H. v. SUSIBA2, barley SUSIBA2; I. b. SPF1, sweet potato SPF1; L. e. hyp,tomato hypothetical proteins; N. t. wrky, tobacoo WRKY protein; R. r.d-i, white broom drought-induced protein.

FIG. 3. Purification of SUSIBA2.

His-tagged SUSIBA2 was subjected to FPLC purification on a Ni affinitycolumn following overexpression of the susiba2 cDNA construct in E.coli. Amino acid sequences obtained by microsequencing of trypticfragments are indicated.

FIG. 4. Binding of SUSIBA2 to Restriction Fragments Containing the SP8aElement.

(A) A portion of the iso1 promoter and restriction fragments spanningthe SP8a element and flanking sequences.

(B) EMSA using the different SP8a-containing restriction fragmentsincubated in the presence (+) or absence (−) of SUSIBA2. The DNA-SUSIBA2complexes are indicated by arrows.

FIG. 5. DNase I Footprint of SUSIBA2 to the iso1 Promoter Region.

(A) The DNA fragment (−736 to −537) was end-labeled and applied toMaxam-Gilbert sequencing. The sequencing reaction was used as markers(M). DNAse I digestion was performed in presence (+) or absence (−) ofSUSIBA2. Positions, relative to the transcriptional start site, in theiso1 promoter are shown on the left. The protected region (−603 to −545)is indicated by an open box. Regions corresponding the SURE-a, SURE-b,and SURE-c sequences are designated as a, b, and c, respectively.

(B) Alignments of sequences from the protected region with the SUREelement from the potato patatin promoter (Grierson et al., 1994).

FIG. 6. Binding of SUSIBA2 to Oligonucleotides Containing the SP8a,SURE, or W box Elements.

(A) The sequence of the three oligonucleotides. The SP8a, SURE and W boxelements are shown in bold.

(B) EMSA using the different oligonucleotides incubated in the presence(+) or absence (−) of SUSIBA2. The DNA-SUSIBA2 complexes are indicatedby arrows.

FIG. 7. Binding Competition Assay for SUSIBA2 with OligonucleotidesContaining the SURE or W box elements.

EMSA using the SURE or W box oligonucleotides incubated in the presence(+) or absence (−) of SUSIBA2, and without (−) or with (SURE or W box) a100-fold molar excess of non-labeled competitor oligonucleotides.

FIG. 8. Temporal Expression Profile for susiba2 and iso1.

(A) RNA gel blot analysis of susiba2 and iso1 gene activity duringbarley endosperm development. Total RNA was isolated from barleyendosperm on different days after pollination (d.a.p.) and used for RNAgel blot analysis. The blots were hybridized with probes specific forsusiba2, iso1, or paz1. The sizes for the hybridizing fragments werearound 2.4 kb for the susiba2 probe, 2.8 kb for iso1 probe, and 1.5 kbfor the paz1 probe.

(B) TLC analysis of endogenous sucrose levels in developing barleyendosperm from different d. a. p. A sucrose extract from 0.2 mg dryweight of developing endosperm from each time point was applied to TLCanalysis.

FIG. 9. Ectopic Expression of susiba2 and iso1 in Barley Leaves.

Barley leaves were treated with exogenous sucrose at the concentrationsindicated. Total RNA was isolated and hybridized to probes specific forsusiba2, iso1, rbcS, and rbcL.

FIG. 10. Effects of the SURE-b ODN Decoy on iso1 Expression inTransformed Barley Endosperm.

(A) Schematic representation of the iso1::gfp construct (upper panel)and the SURE-b ODN decoy (lower panel).

(B) Transient expression assays of GFP fluorescence in transformedbarley endosperm cells. Barley endosperm cells were transformed bymicroprojectile bombardment in absence (−) or presence (+) of the ODNdecoy. For cotransformation, the iso1::gfp construct and ODN decoy weremixed at a molar ratio of 1:100.

(C) Average number of fluorescent foci per plate from nine independenttransformation events.

FIG. 11. Primary Structure Comparison between SUSIBA2, the Rice andWheat SUSIBA2 Orthologs, and SPF1.

(A) Alignment of the amino acid sequences between the conserved WRKYdomains of SUSIBA2, the Rice and Wheat SUSIBA2 Orthologs, and SPF1.

(B) Alignment of the entire amino acid sequence of SUSIBA2 with that ofthe rice SUSIBA2 ortholog. The portion corresponding to the isolatedrice cDNA clone is denoted with a hatched double line under thesequences, with green indicating the aligned regions in (A). The“SUSIBA2-specific” sequence (c.f. FIG. 2) is indicated by a stretch ofblue # signs.

FIG. 12. Schematic Comparison of SUSIBA2 and SUSIBA1.

SUSIBA1 and the C-terminal half of SUSIBA2 share >95% amino acididentity over most of their lengths. The exception is the divergentN-terminus of SUSIBA1. The conserved WRKY domains and the nuclearlocalization signals (NLS) are indicated.

FIG. 13. Model of the Interactions between the SUSIBAs and the iso1Promoter.

SUSIBA2-70 binds to the SURE and W box elements, whereas SUSIBA2-68binds only to the W box. Neither of the SUSIBA2 forms binds to the SP8aelement. Note that we do not know whether SUSIBA2-68 exists in vivo.

FIG. 14. SURE and SURE-like sequences in plant promoters. Alignment ofSURE sequences in promoters of plant genes known to be subjected tosugar induced expression. Selected A and T nucleotides are highlightedin red and green boxes, respectively. SURE-c, SURE-b, and SURE-a referto the SURE sequences of barley iso1 (this work); sbeIIb, barley sbeIIb(Sun et al., 1998); ssI, barley ssI (Gubler et al., 2000); agpaseS,barley agpaseS (Thorbjørnsen et al., 2000); amy, Arabidopsis gene forβ-amylase (Mita et al., 1995); sbeI, maize sbeI (Kim and Guiltinan,1999); sus4, potato gene for sucrose synthase (Fu et al., 1995); vsp,soybean gene for vegetative storage protein (Rhee and Staswick, 1992);PI-II, potato gene for proteinase inhibitor II (Kim et al., 1991); ps20,potato patatin class I gene (Grierson et al., 1994).

EXAMPLES Example 1 Isolation and Characterization of the susiba2 cDNA

Primers were designed for RT-PCR amplification of barley cDNA usingtotal RNA isolated from developing barley endosperm 9 days afterpollination (d.a.p.). Primers combinaed information from sweet potato,cucumber and parsley transcription factor sequences (Ishiguro andNakamura, 1994; Kim et al., 1997; Rushton et al., 1996). Surprisingly,one PCR product of an appropriate size was obtained (data not shown).This is particularly unexpected since given that, for instance, thereare a large number (>70) of WRKY genes in Arabidopsis, and also thatWRKY domains are closely conserved. The choice of developing endospermsmay have contributed to there only being a single PCR product.

The amplified product was used as a probe to screen a 10 d.a.p.endosperm cDNA library and a leaf library. From screening of 1×10⁶p.f.u. 14 positive clones were recovered. All positive clones weresubjected to subcloning, restriction mapping and sequencing analysis.

One clone was completely sequenced on both DNA strands. The length ofthe susiba2 cDNA is 2355 nucleotides, and the open reading frame startsat position 247 and ends at position 1966 (FIG. 1).

The cDNA encodes a 573-amino acids long polypeptide, SUSIBA2, with acalculated molecular mass of 62.2 kDa, and it has 5′ and 3′-untranslatedregions and a poly (A⁺) tail sequences. These findings, together withthe results from RNA gel blot analyses (see below), suggest that thesusiba2 cDNA is full-length. One interesting feature of susiba2 is thatan 18-nucleotides long sequence between nucleotides 1051 and 1068(FIG. 1) is 100% complementary to the gene for human insulin-like growthfactor 1 (IGF1) receptor (nucleotides 173-190).

The deduced amino acid sequence of SUSIBA2 was used in a BLAST search ofthe NCBI databases. Alignment of the 9 most top scoring matches with theSUSIBA2 sequence demonstrated that all ten proteins share a high degreeof similarity and that SUSIBA2 belongs to group 1 of the WRKYsuperfamily of plant transcription factors (FIG. 2).

As would be expected from transcription factors, WRKY proteins containnuclear localization signals (NLSs). The two most common NLSs aremonopartite signals, which are short stretches enriched in basic aminoacids, and bipartite signals, which are composed of two short basicstretches separated by a spacer (Merkle, 2001). The monopartite NLSdepicted for WRKY proteins is conserved in SUSIBA2 (amino acids 328-331;FIG. 2). However, a closer inspection of the sequences suggests that themonopartite NLS in SUSIBA2 and other WRKY proteins might also constitutea bipartite NLS close to the consensus, KR-(24-74)-R..RK, for earlyauxin-inducible proteins (Abel and Theologis, 1995). One putativebipartite NLS in SUSIBA2 is KR-(66)-RK (amino acids 328-397). The otherpossibility is KR-(37)-R.RK (amino acids 328-370) and involves theWRKYGQK sequence itself in the C-terminal WRKY domain. The function ofthe WRKYGQK heptapeptide is unknown and it cannot be ruled out that itparticipates in the nuclear localization process, although a bipartiteNLS arrangement for the N-terminal WRKYGQK sequence is not found. Themost distinguishing feature of SUSIBA2 relative the other WRKY proteinsis the 37-amino acids long insertion following the C-terminal WRKYdomain (amino acids 435-471; FIG. 2). A BLAST search with the insertionsequence did not show any matches in the NCBI databases (however, seebelow), suggesting that SUSIBA2 is a novel type of WRKY protein.

The two WRKY domains (Eulgem et al., 2000) with their zinc finger motifsare highly conserved in SUSIBA2. Also the N-terminal Ser- and Thr-richregions that are present in many WRKY proteins are found in SUSIBA2. Thefunction of these regions is not known but is likely to involveregulation. Ser- and Thr-rich regions are characteristic of activationdomains in transcription factors. It is generally believed that theC-terminal WRKY domain mediates sequence-specific binding to the cognateDNA elements, whereas the N-terminal WRKY domain facilitates DNA bindingor engages in protein-protein interactions (Eulgem et al., 2000). Thezinc fingers in each WRKY domain might be involved in binding to eitherDNA or proteins.

The WRKY class of proteins seem to be specific for the plant kingdom andare best known for their participation in various stress responses andsenescence (Eulgem et al., 2000). Our results demonstrate that theirsphere of activities has to be expanded to also include carbohydrateanabolism. It is probable that the unique properties of SUSIBA2, ascompared to other, hitherto known, WRKY proteins, are attributable toregions outside of the two WRKY domains, where the amino acid sequencesare relatively divergent (FIG. 2).

Example 2 Overproduction of SUSIBA2 in E. coli

The susiba2 cDNA was inserted into a pET expression vector andoverexpressed in E. coli after IPTG induction. The overproduced proteinwas purified by a one-step FPLC on a His-Tag affinity column. Resultsfrom a typical overproduction and purification procedure are shown inFIG. 3.

The purified polypeptides were used for microsequencing, activitycharacterization and antibody production.

The purified protein fraction contained three polypeptides. They wereseparated on SDS-PAGE and subjected to in situ trypsin digestion. Thedigested fragments from each polypeptide were separated on HPLC andapplied to mass-spectrometry analysis. The upper two bands correspondedto polypeptides with molecular masses of around 70 kDa and 68 kDa,respectively, and with the identical sequence LAGGAVP. This sequence isfound in the deduced amino acid sequence of SUSIBA2 (FIG. 1; amino acids235-241). The lower band corresponded to a 28-kDa polypeptide with thesequence of EIAKQA. This sequence matches E. coli trehalose-6-p synthase(amino acids 234-239). This enzyme copurified with the SUSIBA2polypeptides also during repeated runs on the Ni column. It is thereforeconceivable that the copurification was due to an unspecific associationbetween SUSIBA2 and E. coli trehalose 6-P synthase, which may reflect aninteraction of biological significance between SUSIBA2 and barleytrehalose 6-P synthase. A role for trehalose and trehalose-6-phosphatein plant sugar signaling has been proposed (Goddijn and Smeekens, 1998).

Thus purified SUSIBA2 appeared in two forms, SUSIBA2-70 and SUSIBA2-68,of approximately 70 and 68 kDa, respectively (FIG. 3). Possibly,SUSIBA2-68 is a truncated version of SUSIBA2-70. This notion issupported by preliminary results, which indicate that the level ofSUSIBA-70 increases at the expense of SUSIBA2-68 with increasingconcentrations of protease inhibitors.

Since the purified SUSIBA2 still contains the N-terminal His-Tag, itseems less likely that any truncation should be at the N-terminal asthat would leave too few His on SUSIBA2-68 to bind to the Ni column.Alternatively, the size difference between SUSIBA2-70 and SUSIBA-68 isan effect of posttranslational modification. The two SUSIBA2polypeptides were extracted and used for production of polyclonalantibodies.

Example 3 Characterization of SUSIBA2 DNA-Binding Activity

As an initial test of the DNA-binding activity of SUSIBA2, we performedelectrophoresis mobility shift assays (EMSAs) with three overlappingnucleotide fragments from the barley iso1 promoter, all containing theSP8a sequence (FIG. 4).

The 822-bp long SphI/HaeII fragment, encompassing SP8a and flankingsequences, as well as the 437-bp long BspHI/HaeII fragment, containingSP8a and downstream flanking sequence, formed prominent DNA-proteincomplexes with the purified SUSIBA2 fraction. Much to our surprise,however, the 510-bp long SphI/BsaI fragment, containing SP8a andupstream flanking sequence, bound only very poorly to SUSIBA2. Weconcluded that, in fact, the target for SUSIBA2 in the iso1 promoter isnot SP8a but one or more sites downstream of the SP8a element. Fromfurther analysis of the segment downstream of SP8a in the BspHI/HaeIIfragment we found that it harbors the sequence AATACCAAAAAATAATAATAAAA(nucleotides −568 to −545, relative the transcriptinal start site),which shares a high degree of identity with the SURE (sugar responsive)element, first reported by Grierson et al. (1994) from work on thepotato class 1 patatin promoter. To follow up on this observation, wecarried out EMSAs with oligonucleotides consisting of the iso1 SUREsequence. SUSIBA2 exhibited strong binding activity with the SURE probe,whereas binding to the SP8a probe was negligible (FIG. 6).

Since SUSIBA2 is a WRKY protein, it would be expected also to bind tothe W box, the highly conserved binding site for WRKY proteins (Eulgemet al., 2000). A sequence identical to the consensus sequence for the Wbox, TGACT (nucleotides −400 to −396), was located in the iso1 promoter,and the iso1 W box sequence was included in the binding assay. With theW box probe, two DNA-SUSIBA2 complexes could be discerned. Thus, theresults supported our earlier unexpected finding that SUSIBA2 does notbind to the SP8a element, and showed that, instead, it interacts withthe SURE and W box elements.

To further illustrate that SUSIBA2 binds to the SURE element in the iso1promoter, we ran a DNaseI footprinting assay. A 210-bp long NruI/XbaIfragment, encompassing the SURE element, was incubated with purifiedSUSIBA2, followed by DNaseI digestion (FIG. 5).

As expected, the protected region covered the SURE element but also theupstream and downstream flanking sequences were protected. In searchingfor an explanation we found that two additional SURE-like sequences, oneimmediately upstream, CCGAAAAAAACTAAGAAAGACCGATG (nucleotides −603 to−578) and one immediately downstream, AAAAAATAAAGAAAATGAAATC(nucleotides −514 to −493), of the one located initially, are present inthe iso1 promoter. We refer to these SURE sequences as SURE-a, SURE-band SURE-c for the upstream, middle and downstream sequences,respectively. A conceivable scenario is that SUSIBA2 binds to all threeSURE sequences, yielding a protected segment of >110 nucleotides. Thiswould likely explain why the protection continued beyond the 3′ end ofthe restriction fragment (FIG. 5A). As is also suggested by thefootprinting assay, the SUSIBA2-DNA interaction is stronger for SURE-bthan for SURE-a. An attempt to carry out a footprinting assay with the437-bp long BspHI/HaeII fragment (FIG. 4A), covering all three SUREsequences, produced poor quality results. This is not surprising sincethe recommended restriction fragment lengths for DNase I footprinting is250 bp or less.

Having demonstrated that SUSIBA2 binds to the SURE and W box elements inthe iso1 promoter, we were interested in studying the relationshipbetween the two binding activities. To this end we performed acompetition experiment where the same amount of SUSIBA2 was incubatedwith the SURE or W box probe (see FIG. 6), in the presence or absence ofa 100-fold molar excess of non-labeled competitor. The EMSA revealedthat the W box competitor could efficiently outcompete the SURE probefrom SUSIBA2, and that the SURE competitor could outcompete the W boxprobe from the larger of the two SUSIBA2 complexes (FIG. 7). It islogical to assume that the large and small complex contains theSUSIBA2-70 and SUSIBA2-68 polypeptides, respectively. Therefore, weinterpret these results to mean that SUSIBA2-70 binds to both the W boxand the SURE element, whereas SUSIBA2-68 binds only to the W box.

A model depicting the binding properties of SUSIBA2 with respect to theiso1 promoter is shown in FIG. 13. As has been demonstrated above,purified SUSIBA2 binds strongly to the SURE element and the W box butonly poorly to the SP8a motif. Binding to the W box is consistent withSUSIBA2 being a WRKY protein, whereas binding to the SURE elementdisplays a novel feature for a WRKY protein. The results herein suggestthat the extreme C-terminal may be required for SURE-specificinteractions. Another region that merits attention is the insertion inSUSIBA2 right after the C-terminal WRKY domain (FIGS. 2 and 11).

Example 4 Expression of susiba2 and iso1 correlates with Sucrose Levels

In previous investigations we have shown that expression of the barleyiso1 gene is sugar inducible (Sun et al., 1999). In light of thedifferent reports pointing to the involvement of the SURE element insugar signaling (Fu et al., 1995; Grierson et al., 1994; Kim andGuiltinan, 1999; Kim et al., 1991; Li et al, 2002; Mita et al., 1995;Rhee and Staswick, 1992), and the results here, showing the SURE-bindingactivity of the SUSIBA2 transcription factor, it was of interest tostudy the expression pattern of the susiba2 gene. For examination of thetemporal expression, barley endosperms were collected 7 to 27 d.a.p. andtotal RNA was isolated. RNA gel blot analysis was performed withsusiba2- or iso1-specific probes, or with a PCR-amplified probe for thepaz1 gene, encoding the storage protein Z (Sørensen et al., 1989), whichwas included for comparison. The expression level for susiba2 was lowcompared to iso1 but the patterns were similar, with a peak at around 12d.a.p. (FIG. 8A).

A similar time course was obtained also for the barley sbeIIa and sbeIIbgenes (Sun et al., 1998). Expression of the paz1 gene increased up to orbeyond 22 d.a.p. and then sharply declined, consistent with the resultsby Sørensen et al. (1989). To study the correlation between sucrose andsusiba2 expression, we monitored the endogenous sucrose concentrationsduring endosperm development by TLC. The sucrose concentrations weredetermined in seeds from the same batches used for transcript analysis.A comparison between the temporal expression pattern for susiba2 andiso1 transcript accumulation and sucrose levels showed a very goodcorrelation (FIG. 8B).

No susiba2 transcripts could be detected in normal barley leaves.However, ectopic susiba2 expression in leaves was achieved aftersupplying exogenous sugar (FIG. 9A). The SUSIBA2 which was produced insugar-treated leaves where it could be localized to the nucleus (datanot shown).

Ectopic expression of iso1, which agrees with previous results (Sun etal., 1999), was also achieved. As a comparison, the photosynthesis genesrbcS and rbcL, encoding, respectively, the small and large subunit ofRUBISCO, were abundantly expressed in control leaves (0 mM sucrose). Thenuclear rbcS was dramatically downregulated by sucrose while theplastidic rbcL was not notably affected. The expression studies wereextended to the protein level, using the SUSIBA2 antibody in a proteingel blot analysis (FIG. 9B). The SUSIBA2 protein was found in endospermbut not in leaves.

The data from the expression analyses is consistent with a role forSUSIBA2 as a positive regulatory transcription factor for iso1 activity.Experimental evidence supporting this view comes from the antisenseinhibition studies. They also suggest that the expression of the susiba2gene itself is controlled via sugar signaling.

From previous work, we learned that SP8a in the iso1 promoter is anegative regulatory element that recruits a repressor in non-expressingorgans, such as embryos and leaves (Sun et al., 1999). The data from theexpression analyses here, and the affinity of SUSIBA2 for the SUREelement, together strongly suggest that SUSIBA2 binds to the SUREelement(s) in the iso1 promoter as a positive regulator.

Example 5 In vivo Analysis of Transformed Barley Endosperm Demonstratethe Relevance of the SURE Element for iso1 Promoter Activity

To experimentally verify the premise that the SURE-b serves as anactivating element for iso1 expression, we carried out transient assaysin which the transcription of a chimeric iso1-reporter construct wasstudied in transformed barley endosperm. In this experiment, the entire(1.5 kb) iso1 promoter was fused to the gfp reporter gene. Theemployment of the GFP reporter system for analyses of promoter activityhas been successfully used in transformed barley embryos (Ahlandsberg etal., 1999, 2002b). Since the iso1 gene is not expressed in embryos (Sunet al., 1999), a protocol for transformation of barley endosperm wasestablished allowing us to carry out transient assays with gfp reporterconstructs also in transformed barley endosperm.

The activity of the iso1 promoter, manifested as GFP fluorescence, wasfollowed in transformed barley endosperm in the presence or absence of atranscription factor oligodeoxynucleotide (ODN) decoy containing theSURE-b sequence (FIG. 10A).

The ODN decoy approach involves flooding the cells with enoughdouble-stranded decoy to compete for binding of transcription factorswith their consensus sequences in target genes. If present inhigh-enough concentrations, these decoys can negate the ability of thetranscription factor to regulate gene expression. The transcriptionfactor ODN technology is widely used in medical investigations (Mann andDzau, 2000). Here, we choose to adopt the strategy for our studies onbarley endosperm. As is demonstrated in FIG. 10B, the presence of theSURE-b decoy in the endosperm cells blocked most, if not all, iso1promoter activity. As shown in this Figure, the expression of iso1 intransformed barley endosperm was mainly confined to the periphery of theendosperm, towards the aleurone layer. The significance of this finding,if any, is unclear. The endosperms used for transformation were fromseeds 20 d.a.p., which is near maturity. Since programmed cell death ofthe starchy endosperm tissue in barley progresses from the center, theuneven distribution of iso1 activity might indicate that, at 20 d.a.p.,only endosperm cells along the aleuron layer are metabolically viable.Alternatively, the distribution reflects a sugar gradient within theendosperm. The transfer cells, which facilitate transport ofphotosynthate to the endosperm, are located at the basal endosperm,suggesting that, on sucrose medium, the sugar concentration in theendosperm will be highest at the vicinity of the transfer cells (for arecent review on endosperm development, see Olsen, 2001).

The average effect of three independent experiments is shown in FIG.10C.

Example 6 Isolation of cDNA for SUSABA2 Orthologs in Rice and Wheat

In the light of the disclosure above characterising the involvement ofthe SUSIBA2 transcription factor in regulation of iso1 expression inbarley endosperm, it was plausible that SUSIBA2 orthologs should befound in sink tissues also in other plants. The primers used for cloningof the susiba2 cDNA were employed for RT-PCR amplification of total RNAisolated from rice and wheat endosperm. For both species, only one majorRT-PCR product was obtained (data not shown). The PCR products weresubcloned and sequenced.

The deduced amino acid sequences from the PCR products include theregion between the two WRKY domains, and analysis of these segments fromthe rice and wheat sequences show that they share a high degree ofidentity between themselves and with SUSIBA2 (FIG. 11A).

It is notable, apart from the conserved WRKY domains, the overall degreeof identity between SUSIBA2 and the SP8a-binding-SPF1 is rather low(28%; FIG. 11).

The entire sequence for the rice ortholog could be retrieved from theTIGR Rice Genome Project (http://www.tigr.org/tdb/e2k1/osa1/) on anunclassified entry with the temporary name 2017.t00012. Since the openreading frame for the rice clone could be obtained in its entirety, wewere able to compare the complete primary sequence between SUSIBA2 andthe rice ortholog (FIG. 11B). The two proteins are very similar, also inthe extreme C- and N-termini (they share a high degree of amino acididentity (80%) along their entire lengths).

Notably, the “SUSIBA2-specific” C-terminal insertion (see Example 3) ispresent in the rice sequence.

Further investigations have identified SUSIBA2 orthologs also in potatoand Arabidopsis (data not shown).

Example 7 Discussion of Relevance of SURE-Element Binding

The importance of the SURE cis element for sucrose induction of geneactivity in plants was first demonstrated by Grierson et al. (1994) fromwork on the potato patatin promoter, and has been corroborated by alarge number of studies in different plants (Fu et al., 1995; Kim andGuiltinan, 1999; Kim et al., 1991; Li et al, 2002; Mita et al., 1995;Rhee and Staswick, 1992). In some of these studies the assignment of theSURE sequence as a regulatory element was inferred from experimentaldata, i.e. binding assays, whereas in other cases it was based primarilyon computational analyses. Here we show that two (and possibly three)SURE elements are located also in the barley iso1 promoter and that theyprovide binding sites for the SUSIBA2 transcription factor. Its presencein the barley iso1 and maize sbe1 promoter suggests that the SUREelement should also be found in other starch synthesis genes subject tosugar induction. A database search of the proximal and distal promoterregions of sequences revealed that, indeed, SURE-like sequences arepresent in promoters of several genes encoding enzymes involved instarch synthesis. Among those are barley sbeIIb, and the barley genesfor starch SSI (ssI) and the small subunit of AGPase (agpaseS).

Although all three SURE sequences in the barley iso1 promoter, as wellas those identified in other genes, bear a good resemblance to thepatatin SURE sequence in pair-wise comparisons, it is difficult toarrive at a consensus sequence for the SURE element. An alignment of asubset of reported SURE sequences is shown in FIG. 14.

The compilation also includes the SURE sequences from barley ssI andagpaseS. As can be seen, the SURE element is best described as anAT-rich box with the consensus core A/TAAANA, where N denotes anynucleotide. It is similar to the so-called A box response element in thewheat gbss1 promoter (Kluth et al., 2002). A/T-rich protein-bindingsequences are common in both eukaryotic and prokaryotic genes, andcertain protein motifs, such as SPKK, show preference for binding atA/T-rich sites (Churchill and Suzuki, 1989). A/T-rich regions might alsobe expected in some promoters since they facilitate DNA unwinding. Therole of the A/T-rich box in the SURE element remains to be assessed.Certainly, DNA-protein interactions will be affected by the sequencecontext surrounding the A/T-rich region. This is illustrated by thepresence of several A/TAAANA sequences in the DNase I hypersensitiveregion of the iso1 promoter upstream of −608 (FIG. 5).

We noted that, in contrast to the barley sbeIIb promoter, which containsa SURE element (FIG. 14), no SURE-like sequence was found in thepromoter of the barley sbeIIa gene. Both sbeIIb and sbeIIa contain a Wbox (data not shown). We have reported previously (Sun et al., 1998)that expression of barley sbeIIb is endosperm specific while that ofsbeIIa is not. We suggest that the W box in the iso1, sbeIIb and sbeIIapromoters serves as a general activating element while the sugarresponsiveness conferred to iso1 and sbeIIb by the SURE elementscontributes to their endosperm specificity (by the higher sugar levelsin sink tissues as compared to embryos and vegetative tissues). Furthercontrol of endosperm specific expression is probably exerted via bindingof repressor proteins in non-expressing tissues. From earlier work, weconcluded that repressors are recruited to the Sp8a element in thebarley iso1 promoter (Sun et al., 1999), and to the Bbl element in thesecond intron of barley sbeIIb (Ahlandsberg et al., 2002b).

The importance of the SURE element for iso1 expression is demonstratedby the decoy experiment (FIG. 10). The activity remaining in thepresence of the SURE-b decoy could indicate that the decoy did notefficiently trap the SURE-binding activity. However, more likely, itillustrates that elements other than SURE are sufficient to maintain abasic level of iso1 activity. The involvement of the SURE element inregulation of iso1 expression is in line with the ectopic expression ofiso1 in sugar-treated barley leaves (Sun et al., 1999). The emergingtranscription factor ODN technology offers a powerful and convenient wayto assess transcription factor function and involvement of cis elementsboth in vitro and in vivo. The strategy has gained rapid popularity inanimal sciences, e.g. in studies on gene therapy (Mann and Dzau, 2000;Morishita et al., 1998). To our knowledge, the present report representsthe first example of the transcription factor ODN technology in plantbiology.

SUSIBA2 is the first transcription factor known to bind to a SUREelement, and, in fact, it is the first transcription factor reported forregulation of starch synthesis. Although binding of nuclear proteinfractions to the SURE element in the potato patatin (Grierson et al.,1994) and maize sbeI (Kim and Guiltinan, 1999) promoters, to the Sp8asequence in the barley iso1 promoter (Sun et al., 1999), and to the Bblelement in the second intron of the barley sbeIIb gene (Ahlandsberg etal., 2002b) has been documented previously, the interacting transfactors have not been isolated or identified.

Example 8 Down-Regulation of Ectopically Expressed SUSIBA2

To confirm the importance of SUSIBA2 in regulation of starch synthesis,we adopted the antisense oligodeoxynucleotide (ODN) technology [Dagle,J. M. & Weeks, D. L. Differentiation 69, 75-82 (2001)]. The ectopicexpression of iso1 and sbeIIb was followed in intersectedsucrose-treated barley leaves after addition of an 18-mer antisense ODNagainst susiba2. RNA-gel blot analysis revealed that exposure to theantisense ODN suppressed iso1 and sbeIIb expression, providingfunctional evidence that SUSIBA2 is relevant for iso1 and sbeIIbactivities. Expression of sbeIIa or rbcS, encoding the small subunit ofthe Calvin cycle enzyme RUBISCO, were not significantly affected. Theresults of the transient ODN transformation assay demonstrate theapplicability of the antisense ODN technology on intact plant tissues.

Additionally the inventors investigated the effects of the antisense ODNtechnology on starch composition. The antisense susiba2 ODNs lead to asignificant increase in iodine staining, which is indicative of fewer orshorter branches in the starch molecules, consistent with reduced sbeIIband iso1 activities. Also the solubility properties of the starch werealtered, as judged by increased turbidity (results not shown).

Methods used in Examples

Plant Material

Barley (Hordeum vulgare L. cv. Pongo), rice and wheat were grown in soilin a climate chamber under a 16/8 h photoperiod as described by Sun etal. (1998, 1999).

Oligonucleotides

The following oligonucleotide primers were used: Primer 1,5′-CCAAGAAGTTATTACAAGTG-3′ Primer 2, 5′-TGGTTATGTTTTCCTTCGTA-3′ Primer3, 5′-GGAATTCCATATGTCCCCCGCGCGGCTGCC-3′ Primer 4,5′-CGGATCCGGCTGAACTGACTTGTAAC-3′ Primer 5,5′-CCCTCGTGGAAGCAAAACTGTGTTTCTCGC-3′ Primer 6,5′-GCGAGAAACACAGTTTTGCTTCCACGAGGG-3′ Primer 7,5′-GGAAAACCGAAATACCAAAAAATAATAATAAAATAATAAT-3′ Primer 8,5′-ATTATTATTTTATTATTATTTTTTGGTATTTCGGTTTTCC-3′ Primer 9,5′-TCGCTAACCAGTGACTTCCACGTTTCATCATTTATT-3′ Primer 10,5′-AATAAATGATGAAACGTGGAAGTCACTGGTTAGCGA-3′ Primer 11,5′-ATGACTCGAGCAGATTTTGGATTGCTAATGA-3′ Primer 12,5′-ATGACCATGGGCCACCTCGTGTTGGTTCTTCGT-3′Computational Analyses

Searches of the NCBI databases were performed with the BLAST service(http://www.ncbi.nlm.nih.gov/BLAST/). Alignments of nucleotide and aminoacid sequences were carried out using the Clustal W Service at theEuropean Bioinformatics Institute (http://www.ebi.ac.uk/clustalw),except for alignment of sweet potato, cucumber and parsley SPF1 cDNAsequences, which was done with the MacVector® program (Accelrys Inc.,USA), and alignment of the SURE element sequences, which were run on theT-Coffee server (http://www.ch.embnet.org/software/TCoffee.html). Forpresentation purposes, the ALN output from the Clustal W program was fedinto the BOXSHADE server(http://www.ch.embnet.org/software/BOX_form.html.

Isolation of cDNA Clones

Primers 1 and 2 were constructed according to consensus sequences forthe SPF1 and SPF1-like cDNA sequences. Total RNA was isolated fromdeveloping barley, rice and wheat endosperm according to Sun et al.(1999). First-strand cDNAs were produced as described (Frohman et al.,1988) using primer 2. RT-PCR was carried out according to standardprotocols (Sambrook et al., 1989) using primers 1 and 2. The PCRproducts were subcloned and sequenced. The RT-PCR product from barleywas used as a probe to screen a barley cDNA library from developingendosperm 10 days after pollination. Library construction and screening,subcloning, DNA gel-blot analysis and sequence analysis were asdescribed (Sun et al., 1998, 1999).

Overproduction of SUSIBA2 in E. coli, Microsequencing, AntibodyProduction and Protein Gel Blot Analysis

Two primers were designed, primer 3, with an NdeI restriction site, andprimer 4, with a BamHI restriction site. These primers were used for PCRamplification of the susiba2 cDNA from the isolated full-length cDNAclone. The amplified cDNA was inserted into the expression vectorpET-15b (Novagen Inc., Germany) between the NdeI and BamHI sites. Theconstructed plasmid was transformed into E. coli strain BL 21 (DE3).Overexpression was carried out according to the manual provided by themanufacture, except the IPTG induction was performed at 37° C. for 2hrs. The overproduced protein was purified by FPLC™ on a column with aNi⁺ chelating resin. SDS-PAGE was run as described previously (Sun etal. 1997).

Trypsin digestion and microsequencing were done in collaboration withAmersham Pharmacia Biotech., Sweden. Antisera were produced by AgriSeraAB, Sweden after immunizing rabbits with the Coomassie-stained SUSIBA-70and -68 polypeptide bands excised from the SDS gel. Protein gel blotanalysis was performed as before (Sun et al. 1997).

Electrophoresis Mobility Shift Assay (EMSA)

EMSAs were carried out essentially as described by Sun et al. (1999).The competitor probes were prepared by annealing different non-labeledoligonucleotides in 14 mM Tris-HCl (pH 8.0), 7 mM MgCl₂.

DNase I Footprinting

Restriction fragments of the iso1 promoter were singly end-labeled andused together with purified SUSIBA2 and the Sure Track Footpring Kit(Amersham Pharmacia Biotech, Sweden). All procedures were according tothe manual provided by the manufacturer.

RNA Gel Blot Analysis

RNA isolation and RNA gel blot analysis were performed as describedpreviously (Sun et al. 1998, 1999).

Sucrose Isolation and Analysis, and Exogenous Sucrose Induction

Sucrose isolation, TLC and sucrose induction of ectopic gene expressionin barley leaves were carried out as described (Sun et al. 1998, 1999).

Transformation and GFP Assay of Barley Endosperm

The 1504-bp long promoter region of the iso1 gene was PCR-amplifiedusing primers 11 and 12 and fused to the gfp plasmid pN1473GFP asdescribed by Ahlandsberg et al. (1999, 2002a), yielding the constructp48. The ODN SURE-b decoy was generated by annealing primers 7 and 8 in14 mM Tris-HCl (pH 8.0), 7 mM MgCl₂. Transformation of barley endospermby microprojectile bombardment, using a DuPont PDS 1000 He BiolisticDelivery System (Bio-Rad Laboratories, Hercules, Calif., USA), andtransient assay of GFP fluorescence were performed as described byAhlandsberg et al. (2002a) with the following exceptions. Barleycaryopses of 19-22 d.a.p were bisected longitudinally and placedendosperm side up on DG3B medium, containing Murashige and Skoog medium(Murashige and Skoog, 1962) supplemented with 100 g l⁻¹ sucrose, 1.0 mgl⁻¹ thiamine-HCl, 0.25 g l-1 myo-inositol, 1.0 g l⁻¹ casein hydrolase,and 0.69 g l⁻¹ proline solidified by 5.0 g l⁻¹ Gelrite (Duchefa,Haarlem, The Netherlands), for two hours prior bombardment. Six bisectedcaryopses were placed in the center of a 20×90 mm Petri dish with DG3Bmedium, 13 cm below the stopping screen, and bombarded once using a 7.6MPa rupture disc. After bombardment, the caryopses were kept for 48hours in darkness on plates before GFP fluorescence assays.

Accession Numbers

GenBank accession numbers referred to in this work are: Arabidopsis genefor β-amylase, S77076; Arabidopsis hypothetical protein, T08930;Arabidopsis WRKY transcription factor 20, Q93WV0; Arabidopsis putativeprotein, NP567752; Arabidopsis unknown protein, AAK76566; barleyagpaseS, AJ239130; barley iso1, AF142589; barley sbeIIb, AF064563;barley ssI, AF234163; barley WRKY proteins implicated in droughttolerance, BM816210 and BM816211; E. coli trehalose-6-P synthase,S33584; human IGF1 receptor, NM_(—)000875; maize sbeI, AF072724; potatopatatin class I gene, M18880; potato gene for proteinase inhibitor II,X04118; Potato gene for sucrose synthase, U24087; soybean gene forvegetative storage protein, M76980; sweet potato SPF1, S51529; tobaccoWRKY protein, BAB61056; tomato hypothetical proteins, CAC36397 andCAC36402; white broom drought induced protein, AAL32033.

After the priority date of the present application, the followingsequences of the present invention were deposited:

Hordeum vulgare SUSIBA2 (susiba2) mRNA, complete cdsgi|34329336|gb|AY323206.1|[34329336]; Oryza sativa SUSIBA2-like proteinmRNA, partial cds; gi|34329334|gb|AY324393.1|[34329334]; Triticumaestivum SUSIBA2-like protein mRNA, partial cdsgi|34329332|gb|AY324392.1|[34329332].

SUSIBA2 [Hordeum vulgare] gi|34329337|gb|AAQ63880.1|[34329337];

SUSIBA2-like protein [Oryza sativa]

gi|34329335|gb|AAQ63879.1|[34329335];

SUSIBA2-like protein [Triticum aestivum]

gi|34329333|gb|AAQ63878.1|[34329333].

Antisense Oligodeoxynucleotide (ODN) Technology

Barley (Hordeum vulgare cv Pongo) plants were grown in soil in a climatechamber as described by Sun et al. (1998, 1999). The plants were usedafter 12-h darkness. Barley leaves were detached and incubated with 200mM Suc solution in presence of 5 μM of antisense ODN or sense ODN. One18-mer antisense ODN (oligonucleotide 1) against barley susiba2 cDNAsequences was used and three of sense ODN of the same length(oligonucleotides 2-4) were used as controls. The sequences ofoligonucleotides 1-4 were as follows: 1, 5′-CGCGGGGGACATGGCCTT-3′; 2,5′-AAGGCCATGTCCCCCGCG-3′; 3, 5′-CCAGACATGCTGCCTTCG-3′; 4,5′-CCTGCTATGAGTGATCTA-3′. After 24 h-incubation in darkness, the barleyleaves were harvested and stored at −80° C. until further use.

Sequence Annex

Rice susiba2 cDNA CGTTCGCTTGATGGTCAGATTACTGAAGTGGTTTATAAAGGGCGTCACAATCACCCTAAGCCCCAACCCAATAGGAGGCTGTCTGCCGGTGCAGTTCCTCCAATCCAGGGTGAAGAAAGATATGATGGTGTGGCAACTACTGATGACAAATCTTCAAATGTTCTTAGCATTCTTGGTAATGCAGTACATACAGCTGGTATGATTGAGCCTGTTCCAGGCTCAGCTAGTGATGATGACAATGATGCCGGAGGAGGGAGACCTTACCCTGGAGATGATGCTGTTGAGGATGATGATTTAGAGTCAAAACGAAGGAAAATGGAATCTGCTGCTATTGATGCTGCTTTGATGGGCAAGCCTAACCGTGAGCCTCGTGTTGTAGTACAAACGGTTAGTGAGGTTGACATCTTGGATGATGGGTACCGCTGGCGCAAGTATGGCCAGAAAGTAGTTAAAGGAAACCCCAATCCACGGAGTTACTACAAGTGCACAAATACAGGATGCCCAGTCAGGAAGCATGTTGAGAGAGCATCACATGATCCAAAATCAGTCA TAACAACATACGAAGG(the rice ortholog sequence can be retrieved from the TIGR Rice GenomeProject (http://www.tigr.org/tdb/e2k1/osa1/) on an unclassified entrywith the temporary name 2017.t00012)

Wheat susiba2 cDNA ACAAGTGCACACATCCTAATTGTGAAGTAAAAAAGCTATTGGAGCGTGCGGTTGATGGTCTGATCACGGAAGTTGTCTATAAGGGGCGCCATAATCATCCTAAGCCCCAGCCTAATAGGAGGTTAGCTGGTGGTGCAGTTCCTTCGAACCAGGGTGAAGAACGATATGATGGTGCGGCAGCTGCTGATGATAAATCTTCCAATGCTCTTAGCAACCTTGCTAATCCGGTAAATTCGCCTGGCATGGTTGAGCCTGTTCCAGTTTCAGTTAGTGATGATGACATAGATGCTGGAGGTGGAAGACCCTACCCTGGGGATGATGCTACAGAGGAGGAGGATTTAGAGTTGAAACGCAGGAAAATGGAGTCTGCAGGTATTGATGCTGCTCTGATGGGTAAACCTAACCGTGAGCCCCGTGTTGTCGTTCAAACTGTAAGTGAGGTTGACATCTTGGATGATGGGTATCGTTGGCGGAAATATGGACAGAAAGTTGTCAAAGGAAACCCCAATCCACGGAGTTACTACAAATGCACAAGCACAGGATGCCCTGTGAGGAAGCATGTTGAGAGAGCATCGCATGATCCTAAATCAGTGATAACAACGTACGAAGGAAAACATAACCA

Wheat susiba2 Peptide PRSYYKCTHPNCEVKKLLERAVDGLITEVVYKGRHNHPKPQPNRRLAGGAVPSNQGEERYDGAAAADDKSSNALSNLANPVNSPGMVEPVPVSVSDDDIDAGGGRPYPGDDATEEEDLELKRRKMESAGIDAALMGKPNREPRVVVQTVSEVDILDDGYRWRKYGQKVVKGNPNPRSYYKCTSTGCPVRKHVERASHDPK SVITTYE

REFERENCES

-   Ahlandsberg, S. Sathish, P., Sun, C. and Jansson, C. (1999). Green    fluorescent protein as a reporter system in the transformation of    barley cultivars. Physiol. Plant. 107, 194-200.-   Ahlandsberg, S. Sathish, P., Sun, C. and Jansson, C. (2002a). A set    of useful monocotyledon transformation vectors. Biotech. Lett. 23,    1871-1875.-   Ahlandsberg, S., Sun, C., and Jansson, C. (2002b). An intronic    element directs endosperm-specific expression of the sbeIIb gene    during barley seed development. Plant Cell Rep. 20, 864-868.-   Abel, S., and Theologis, A. (1995). A polymorphic bipartite motif    signals nuclear targeting of early auxin-inducible proteins related    to PS-IAA4 from pea (Pisum sativum). Plant J. 8, 87-96.-   Bae, J. M., and Liu J. R. (1997). Molecular cloning and    characterization of two novel isoforms of the small subunit of    ADP-glucose pyrophosphorylase from sweet potato. Mol. Gen. Genet.    254, 179-185.-   Ball, S., van de Wal, M. H. B. J., and Visser, R. G. F. (1998).    progress in understanding the synthesis of amylose. Trends Plant    Sci. 3, 462-467.-   Buléon, A., Colonna, P., Planchot, V. and Ball, S. (1998). Starch    granules: structure and biosynthesis. Int. J. Biol. Macromol. 23,    85-112.-   Carlson, M. (1999). Glucose repression in yeast. Curr. Opin.    Microbiol. 2, 202-207.-   Churchill, M. E. A., and Suzuki, M. (1989). ‘SPKK’ Motifs Prefer to    Bind to DNA at A/T-rich Sites. EMBO J. 8, 4189-4195.-   Eulgem, T., Rushton, P. J., Robatzek, S., and Somssich, I. E.    (2000). The WRKY superfamily of plant transcription factors. Trends    Plant Sci. 5, 199-206.-   Frohman, M. A., Dush, M. K., Martin, G. R. (1988). Rapid production    of full-length cDNAs from rare transcripts: Amplification using a    single gene-specific oligonucleotide primer. Proc. Natl. Acad. Sci.    USA 85, 8998-9002.-   Fu, H., Kim, S. Y., and Park, W. D. (1995). High-level tuber    expression and sucrose inducibility of a potato sus4 sucrose    synthase gene require 5′ and 3′flanking sequences and the leader    intron. Plant Cell 7, 1387-1394.-   Giuliano, G., Pichersky, E., Malik, V. S., Timko, M. P., Scolnic, P.    A., and Cashmore, A. R. (1988). An evolutionary conserved protein    binding sequence upstream of a plant light-regulated gene. Proc.    Natl. Acad. Sci. USA 85, 7089-7093.-   Goddijn, O., and Smeekens, S. (1998). Sensing trehalose biosynthesis    in plants. Plant J., 14, 143-146.-   Grierson, C., Du, J.-S., de Torres Zabalau, M., Beggs, K., Smith,    C., Holdsworth, M., and Bevan, M. W. (1994). Separate cis sequences    and trans factors direct metabolic and developmental regulation of a    potato tuber storage protein gene. Plant J. 5, 815-826.-   Gubler, F., Li, Z., Fieg, S., Jacobsen, J. V., and Morell, M. K.    (2000). Cloning and characterization of a starch synthase I gene    (accession No. AF234163) from barley. Plant Physiol. 122, 1459.-   Halford, N. G., and Hardie, D. G. (1998). SNF1-related protein    kinases: Global regulators of carbon metabolism in plants? Plant    Mol. Biol. 37, 735-748.-   Hardie, D. G., Carling, D., and Carlson, M. (1998). The    AMP-activated/SNF1 protein kinase subfamily: Metabolic sensors of    the eukaryotic cell? Annu. Rev. Biochem. 67, 821-855.-   Hattori, T., Nakagawa, S., and Nakamura, K. (1990). High-level    expression of tuberous root storage protein genes of sweet potato in    stems of plantlets grown in vitro on sucrose medium. Plant Mol.    Biol. 14, 595-604.-   Ishiguro, S., and Nakamura, K. (1992). The nuclear factor SP8BF    binds to the 5′-upstream regions of three different genes coding for    major proteins of sweet potato tuberous roots. Plant Mol. Biol. 18,    97-108.-   Ishiguro, S., and Nakamura, K. (1994). Characterization of a cDNA    encoding a novel DNA-binding protein, SPF1, that recognizes SP8    sequences in the 5′ upstream regions of genes coding for sporamin    and β-amylase from sweet potato. Mol. Gen. Genet. 244, 563-571.-   Khoshnoodi, J., Larsson, C.-T., Larsson, H., and Rask, L. (1998).    Differential accumulation of Arabidopsis thaliana Sbe2.1 and Sbe2.2    transcripts in response to light. Plant Sci. 135, 183-193.-   Kim, S.-R., Costa, M. A., and An, G. (1991). Sugar response element    enhances wound response of potato proteinase inhibitor II promoter    in transgenic tobacco. Plant Mol. Biol. 17, 973-983.-   Kim, D.-J., Smith, S. M., and Leaver, C. J. (1997). A cDNA encoding    a putative SPF1-type DNA-binding protein from cucumber. Gene 185,    265-269.-   Kim, K.-N., and Guiltinan, M. J. (1999). Identification of    cis-acting elements important for expression of the starch-branching    enzyme I gene in maize endosperm. Plant Physiol. 121, 225-236.-   Kluth, A., Sprunck, S., Becker, D., Lörz, H., and Lütticke, S.    (2002). 5′ deletion of a gbsss1 promoter region from wheat leads to    changes in tissue and developmental specificities. Plant Mol. Biol.    49, 669-682.-   Kossman, J., Visser, R. G. F., Müller-Röber, B., Willmitzer, L., and    Sonnewald, U. (1991). Cloning and expression analysis of a potato    cDNA that encodes branching enzyme: evidence for coexpression of    starch biosynthetic genes. Mol. Gen. Genet. 230, 39-44.-   Li, X., Xing, J., Gianfagna, T. J., and Janes, H. W. (2002). Sucrose    regulation of ADP-glucose pyrophosphorylase subunit genes transcript    levels in leaves and fruits. Plant Sci. 162, 239-244-   Maeo, K., Tomiya, T., Hayashi, K., Akaike, M., Morikami, A.,    Ishiguro, S., Nakamura, K. (2001). Sugar-responsible elements in the    promoter of a gene for β-amylase of sweet potato. Plant Mol. Biol.    46, 627-637.-   Mann, M. J., and Dzau, V. J. (2000). Therapeutic applications of    transcription factor decoy oligonucleotides. J. Clin. Invest. 106,    1071-1075.-   Merkle, T. (2001). Nuclear import and export of proteins in plants:    a tool for the regulation of signaling. Planta 213, 499-517.-   Mita, S., Suzuki-Fujii, K., and Nakamura, K. (1995). Sugar-inducible    expression of a gene for β-amylase in Arabidopsis thaliana. Plant    Physiol. 107, 895-904.-   Morishita, R., Higaki, J., Tomita N., and Ogihara T. (1998).    Application of transcription factor “decoy” strategy as means of    gene therapy and study of gene expression in cardiovascular disease.    Circ. Res. 82, 1023-1028.-   Murashige, T., and Skoog, F. (1962). A revised medium for rapid    growth and bio assays with tobacco tissue cultures. Physiol. Plant.    15, 473-497.-   Müller-Röber, B. T., Kossman, J., Hannah, L. C., and Willmitzer, L.    (1990). One of two different ADP-glucose pyrophosphorylase genes    from potato responds strongly to elevated levels of sucrose. Mol.    Gen. Genet. 224, 136-146.-   Myers, A. M., Morell, M. K., James, M. G., and Ball, S. G. (2000).    Recent progress toward understanding biosynthesis of the amylopectin    crystal. Plant Physiol. 122, 989-997.-   Nakamura, Y. (1996). Some properties of starch debranching enzymes    and their possible role in amylopectin biosynthesis. Plant Sci. 121,    1-18.-   Nakamura, Y. (2002). Towards a better understanding of the metabolic    system for amylopectin biosynthesis in plants: rice endosperm as a    model tissue. Plant Cell. Physiol. 43, 718-725.-   Olsen, O.-A. (2001). Endosperm development: Cellularization and cell    fate specification. Annu. Rev. Plant Physiol. Plant Mol. Biol. 52,    233-267.-   Ozturk, Z. N., Talamé, V., Deyholos, M., Michalowski, C. B.,    Galbraith, D. W., Gozukirmizi, N., Tuberosa, R., and Bohnert, H. J.    (2002). Monitoring large-scale changes in transcript abundance in    drought- and salt-stressed barley. Plant Mol. Biol. 48, 551-573.-   Rhee, Y., and Staswick, P. E. (1992). Nucleotide sequence of a    soybean vegetative storage protein vspB gene. Plant Physiol. 98,    794-795.-   Rolland, F., Moore, B., and Sheen, J. (2002). Sugar sensing and    signaling in plants. Plant Cell S185-S205.-   Rook, F., Corke, F., Card, R., Munz, G., Smith, C., and Bevan, M. W.    (2001). Impaired sucrose-induction mutants reveal the modulation of    sugar-induced starch biosynthetic expression by abscisic acid    signaling. Plant J. 26, 421-433.-   Rushton, P. J., Torres, J. T., Parniske, M., Wernert, P., Hahlbrock,    K., and Somssich, I. E. (1996). Interaction of elicitor-induced    DNA-binding proteins with elicitor response elements in the    promoters of parsley PR1 genes. EMBO J. 15, 5690-5700.-   Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989). Molecular    Cloning. New York, Cold Spring Harbor Laboratory Press.-   Sheen, J., Zhou, L., and Jang, J. C. (1999). Sugars as signaling    molecules. Curr. Opin. Plant Biol, 2, 410-418.-   Smeekens, S. (2000). Sugar-induced signal transduction in plants.    Annu. Rev. Palnt Physiol. Plant Mol. Biol. 51, 49-81.-   Smith, A. M. (2001). The biosynthesis of the starch granule.    Biomacromol. 2, 335-341.-   Sun, C., Sathish, P., Ahlandsberg, S., Deiber, A., and    Jansson, C. (1997) Identification of four starch-branching enzymes    in barley endosperm: partial purification of forms I, IIa and IIb.    New Phytol. 137, 215-222.-   Sun, C., Sathish, P., Ahlandsberg, S., and Jansson, C. (1998). The    two genes. encoding starch-branching enzyme IIa and IIb are    differentially expressed in barley. Plant Physiol 118, 37-49.-   Sun, C., Sathish, P., Ahlandsberg, S., and Jansson, C. (1999).    Analyses of isoamylase gene activity in wild-type barley indicate    its involvement in starch synthesis. Plant Mol. Biol. 40, 431-443.-   Sørensen M. B., Cameron-Mills, V., and Brandt, A. (1989).    Transcriptional and post-transcriptional regulation of gene    expression in developing barley endosperm. Mol. Gen. Genet. 217,    195-201.-   Thorbjørnsen, T., Villand, P., Ramstad, V. E., Kleczkowski, L. A.,    Olsen, O.-A., and Opsahl-Ferstad, H.-G. (2000). Nucleotide sequence    of the ADP-glucose pyrophosphorylase promoter (accession No.    AF239130) of barley a gene involved in endosperm starch formation.    Plant Physiol. 122, 1459.-   Visser, R. G. F., Stolte, A, and Jacobsen, E. (1991). Expression of    a chimaeric granule-bound starch synthase-GUS gene in transgenic    potato plants. Plant Mol. Biol. 17, 691-699.-   Zourelidou, M., de Torres-Zabala, M., Smith, C., and Bevan, M. W.    (2002). Storekeeper defines a new class of plant-specific    DNA-binding proteins and is a putative regulator of patatin    expression. Plant J. 30, 489-497.

1. An isolated nucleic acid which comprises a nucleotide sequence whichencodes a sugar-signalling transcription factor which is capable ofactivating a promoter of a gene encoding an enzyme involved in thesynthesis or deposition of starch.
 2. A nucleic acid as claimed in claim1 wherein the transcription factor is a WRKY protein which is capable ofactivating the promoter within a plant in response to sugar levels inthe plant
 3. A nucleic acid as claimed in claim 2 wherein the promotercomprises at least one SURE element and\or W box element to which thetranscription factor binds
 4. A nucleic acid as claimed in claim 3wherein the promoter is selected from the group consisting of iso1,sbe1, sbeIIb, ssI, and agpaseS.
 5. A nucleic acid as claimed in claim 1wherein the nucleotide sequence is a susiba2 nucleotide sequence which:(i) encodes the SUSIBA2 polypeptide given in FIG. 1, or (ii) encodes avariant SUSIBA2 polypeptide which is a variant of the SUSIBA2 amino acidsequence given in FIG. 1 and which shares at least about 50%, 60%, 70%,80% or 90% identity therewith.
 6. A nucleic acid as claimed in claim 5wherein the nucleotide sequence: (i) consists of the barley susiba2coding sequence given in FIG. 1 or one which is degenerativelyequivalent thereto, (ii) comprises a wheat or rice susiba2 codingsequence given in the Sequence Annex, or one which is degenerativelyequivalent to either.
 7. A nucleic acid as claimed in claim 5 whereinthe susiba2 nucleotide sequence encodes a derivative of a susiba2 codingsequence selected from the group consisting of the barley susiba2 shownin FIG. 1 or a sequence which is degeneratively equivalent thereto, awheat susiba2 coding sequence or a sequence which is degenerativelyequivalent thereto, a rice susiba2 coding sequence or a sequence whichis degeneratively equivalent thereto by way of addition, insertion,deletion or substitution of one or more codons.
 8. A nucleic acid asclaimed in claim 5 wherein the susiba2 nucleotide sequence consists ofan allelic or other homologous or orthologous variant of the barleysusiba2 coding sequence given in FIG.
 1. 9. An isolated nucleic acidwhich comprises a nucleotide sequence which is the complement of thetranscription factor-encoding nucleotide sequence of claim
 5. 10. Anisolated nucleic acid for use as a probe or primer, said nucleic acidhaving a distinctive sequence of at least about 16-24 nucleotides inlength, which sequence is present in FIG. 1 or a sequence which isdegeneratively equivalent thereto, or the complement of either.
 11. Anisolated nucleic acid as claimed in claim 10 wherein the distinctivesequence encodes all or part of the SUSIBA2-specific sequence:ppmknvvhqinsnmpssiggmmracearnytnqysqaa.
 12. A process for producing anucleic acid as claimed in claim
 7. 13. A method for identifying orcloning a nucleic acid as claimed in claim 8, which method employs anucleic acid probe or primer having a distinctive sequence of at leastabout 16-24 nucleotides in length, which sequence is present in FIG. 1or a sequence which is degeneratively equivalent thereto, or thecomplement of either.
 14. A method as claimed in claim 13, which methodcomprises the steps of: (a) providing a preparation of nucleic acid froma plant cell; (b) providing said nucleic acid probe or primer, (c)contacting nucleic acid in said preparation of step (a) with said probeor primer under conditions for hybridisation, and, (d) identifyingnucleic acid in said preparation which hybridises with said nucleic acidmolecule.
 15. A method as claimed in claim 13, which method comprisesthe steps of: (a) providing a preparation of nucleic acid from a plantcell; (b) providing a pair of nucleic acid molecule primers suitable forPCR, (c) contacting nucleic acid in said preparation with said primersunder conditions for performance of PCR, (d) performing PCR anddetermining the presence or absence of amplified PCR product.
 16. Arecombinant vector which comprises the nucleic acid of claim
 1. 17. Avector as claimed in claim 16 wherein the nucleic acid is operablylinked to a promoter for transcription in a host cell, wherein thepromoter is optionally an inducible promoter.
 18. A vector as claimed inclaim 16 which is a plant vector.
 19. A method which comprises the stepof introducing the vector of claim 16 into a host cell, and optionallycausing or allowing recombination between the vector and the host cellgenome such as to transform the host cell.
 20. A host cell containing ortransformed with a heterologous vector of claim
 16. 21. A method forproducing a transgenic plant, which method comprises the steps of: (a)providing the host cell of claim 20, (b) regenerating a plant from thetransformed plant cell.
 22. A transgenic plant which is obtainable bythe method of claim 17, or which is a clone, or selfed or hybrid progenyor other descendant of said transgenic plant, which in each caseincludes a heterologous nucleic acid which comprises a nucleotidesequence encoding a sugar-signalling transcription factor which iscapable of activating a promoter of a gene encoding an enzyme involvedin the synthesis or deposition of starch.
 23. A transgenic plant asclaimed in claim 22 which is a seed crop plant.
 24. A part of propagulefrom a plant as claimed in claim 22, which includes a heterologousnucleic acid which comprises a nucleotide sequence encoding asugar-signalling transcription factor which is capable of activating apromoter of a gene encoding an enzyme involved in the synthesis ordeposition of starch, said plant optionally being a seed crop plant. 25.An isolated polypeptide sugar-signalling transcription factor which isencoded by the nucleotide sequence of claim
 1. 26. A polypeptide asclaimed in claim 25 which is the SUSIBA2 polypeptide shown in FIG. 1.27. (canceled)
 28. A method for activating the promoter of a geneencoding an enzyme involved in the synthesis or deposition of starch ina plant, wherein the promoter is activated by a sugar-signallingtranscription factor, which method comprises the step of causing orallowing expression of a heterologous nucleic acid as claimed in claim 1within the cells of the plant, thereby expressing the transcriptionfactor therein.
 29. A method as claimed in claim 28 which is preceded bythe earlier step of introduction of the heterologous nucleic acid into acell of the plant or an ancestor thereof.
 30. A method for modulatingthe activity of a promoter of a gene encoding an enzyme involved in thesynthesis or deposition of starch in a plant, wherein the promoter isactivated by a sugar-signalling transcription factor, which methodcomprises any of the following steps of: (i) introducing all or part ofa nucleic acid as claimed in claim 9 in the plant such as to reducetranscription factor expression by an antisense ODN mechanism; (ii)causing or allowing transcription from part of a nucleic acid whichcomprises a nucleotide sequence encoding a sugar-signallingtranscription factor which is capable of activating a promoter of a geneencoding an enzyme involved in the synthesis or deposition of starchsuch as to reduce transcription factor expression by co-suppression;(iii) providing a nucleic acid encoding a ribozyme specific for anucleic acid which comprising a sequence encoding a sugar-signallingtranscription factor which is capable of activating a promoter of a geneencoding an enzyme involved in the synthesis or deposition of starch,(iv) providing a double-stranded RNA which comprises an RNA sequenceencoding part of a sugar-signalling polypeptide, which is optionally asiRNA duplex consisting of between 20 and 25 base pairs.
 31. A method ofproducing modified starch anabolism activity in plant comprising use ofa method of claim 28, and optionally recovering starch from the plant.32. A method of binding, activating, or identifying a promoter whichincludes at least one SURE element and\or W box element, which methodemploys the step of contacting said promoter with a polypeptide of claim25.
 33. A method of investigating or confirming whether a cis promoterelement is present in a plant transcription factor consensus sequence ina target gene promoter, the method comprising: (i) observing theexpression of a reporter gene operably linked to the promoter in a plantcell in which the transcription factor is present, (ii) introducing intothe plant cell a double stranded oligodeoxynucleotide (ODN) decoycorresponding to the promoter element into the cell, (iii) observing theexpression of the reporter gene in the presence of the ODN decoy,wherein a reduction in expression from (i) to (iii) confirms that theplant transcription factor binds the promoter element. 34-37. (canceled)